Introduction Splenic B-cell lymphomas, (SLs, total incidence ~1:100,000/year) comprise four distinct indolent B-cell non-Hodgkin lymphomas. Three predominantly involve the red pulp of the spleen ...(classical hairy cell leukemia (HCL), hairy cell leukemia variant (HCLv) and splenic diffuse red pulp small B-cell lymphoma (SDRPL)) and the fourth predominantly involves the white pulp (splenic marginal zone lymphoma (SMZL)). HCL is usually diagnosed by detection of an activation mutation in BRAF and responds well to treatment. We focused our study on the remaining three diseases, which have overlapping clinical presentations with only subtle differences in their morphology and phenotypes, but have considerable differences in prognosis, response to treatment and outcome. The white pulp lymphoma SMZL generally has good treatment responses and longer survival, whereas the red pulp SLs, HCLv and SDRPL are less responsive to treatments with shorter survival. Cells of the tumor microenvironment (TME) may have distinct characteristics between these three diseases. We hypothesized that TME innate immune cells from red pulp SLs (HCLv, and SDRPL) would be similar to each other and distinct from the white pulp SL, SMZL.In this study, we report that natural killer (NK) cells, which are important innate immune cells critical for immune surveillance, vary in proportions among red and white pulp SLs. Methods We studied splenectomy specimens from patients with SMZL (n=8), HCLv (n=8), SDRPL (n=7), and controls (healthy, post-trauma, n=8). Cryo-preserved bulk splenic suspension cell isolates from fresh (<4 hour) surgical specimens and intact formalin-fixed paraffin embedded (FFPE) tissue sections were used. Human specimen collection and usage was conducted with written informed consent after approval of the Institutional Research Subjects Review Board. FFPE tissue sections were studied by immunohistochemical (IHC) staining and splenic suspension cells were analyzed by high-parameter fluorescence flow cytometry using a Cytek Aurora spectral flow cytometer with a 25-color myeloid and a 28-color NK/B-cell panel. Results IHC characterization showed an extensive network of innate immune cells as reflected by CD163 + macrophages. Using high-parameter flow cytometry, we focused on innate immune cells in the non-B- non-T-cell (CD3 -CD19 -) population. The average proportions of this population were: NK cells 25-36%, monocyte/macrophages 20-34%, dendritic cells 5-11%, and granulocytes 2%. Among the patient groups, the median percentage of NK cells was greater for controls (34%) and SMZL (35%) as compared to red pulp lymphomas (HCLv (28%), SDRPL (24%)). Next, we examined NK subtypes: 1) cytotoxic NK cells (CD56 +CD16 +), 2) cytokine-secreting weakly cytotoxic NK cells (CD56 +CD16 -), and 3) undefined NK cells (CD56 -CD16 +) as a median percentage of total NK cells. The major subtype for control and SMZL was CD56 +CD16 - with values of 58 and 44%, respectively. This subtype comprises only 38%-40% of red pulp SLs. Proportionally the CD56 +CD16 + subtype was the largest for red pulp SLs at 52-57% as compared to 30% for control and 38% for SMZL. The remaining CD56 -CD16 + NK cell subtype was generally higher among red pulp SLs (HCLv (9%), SDRPL (12%)) as compared to control (6%) and SMZL (4%) patients. The increase in cytotoxic CD56 +CD16 + subtype among red pulp SLs was confirmed by measuring the percentage of granzymeB +perforin + cells among mature cytotoxic NK cells (CD56 +CD16 +/ CD57 +NKG2a -). SLs had higher proportions of this NK phenotype (78-86% compared to control 69%) and red pulp SLs had the most granzymeB +perforin + proportions with 82 and 86% for HCLv and SDRPL, respectively. Discussion In summary, spleens from patients with SMZL had similar percentages of total NK cells to controls, with lower percentages in red pulp SLs. All SLs had a higher percentage of cytotoxic NK cells than control, with the red pulp SLs having the most. These data suggest that monoclonal antibody treatments targeting SL antigen, such as anti-CD20, may be effective by activating cytotoxic NK cell to target lymphoma cells by antibody-dependent cellular cytotoxicity. Thus, uncovering the characteristics of the TME may aid the development of non-invasive diagnostic procedures and/or precision therapies.
Antigen-specific T cells demonstrate several potent effector functions during immune responses. Direct killing of infected cells is crucial for clearing viruses and other intracellular pathogens, but ...it has been difficult to measure the frequency of cytolytic cells. We have now developed a single-cell assay to measure the number of cytotoxic cells in a population, using a herpes simplex virus amplicon vector to express Escherichia coli β-galactosidase in mouse or human target cells, and an Elispot to detect release of β-galactosidase from killed target cells. This antigen-specific, perforin-dependent Lysispot assay has been combined with a cytokine Elispot in a two-color assay to confirm that cytotoxicity and interferon-γ secretion are regulated independently. The simultaneous enumeration of cytokine-secreting and cytotoxic cells should be invaluable for ex vivo analysis of immune responses during infection and autoimmunity.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Although T cell effector subsets, defined by cytokine patterns, have been recognized for more than 20 years, the functional cytokine expression patterns in vivo are still in considerable doubt, ...particularly for human T cells. At least three new subsets have been recently identified, but the committed cytokine pattern of a T cell (e.g., Th1 cells produce IL-2, interferon-gamma, and lymphotoxin) may differ from the expression pattern of one cell on one occasion, which may be a subset of its full potential. Recent advances in flow cytometry allowed detailed cytokine patterns of antigen-stimulated cells to be identified directly ex vivo. These patterns are clearly more diverse than the major subsets identified as committed phenotypes. Additional contributions to diversity may include new committed subsets, random expression of only part of the committed pattern, and modification of the expression patterns by cytokines and other mediators.
Primed CD4 T cells may develop into effector T cells such as Th1 and Th2, or remain uncommitted as Th primed precursor (Thpp) cells that can subsequently differentiate into Th1 and Th2 cells. ...Although mouse Thpp-like cells have also been identified among spleen and particularly lymph node cells, further characterization of these cells has been difficult without a defining cell surface marker. Using Affymetrix GeneChips followed by FACS analysis, we found that in vitro-derived Thpp cells expressed CD73 but not Ly-6A/E, whereas Th1 and Th2 cells showed the reciprocal pattern. CD73+ Ly6A/E- memory CD4 T cells were identified in normal C57BL/6 mice, and the proportion of these cells was highest in lymph nodes, lower in spleens, and lowest in the lungs. These cells produced IL-2 and MIP-1alpha, but much less IL-4 and IFN-gamma than CD73- Ly6A/E+ cells. Similar results were obtained with additional Ly-6.2 mouse strains, but not Ly-6.1 strains. Restimulation of Thpp-like CD73+ Ly-6A/E- cells in Th1- or Th2-polarizing conditions induced differentiation into populations producing mainly IFN-gamma or mainly IL-4, respectively. In contrast, the effector-like CD73- Ly-6A/E+ population was more committed, and continued to produce both IL-4 and IFN-gamma in both conditions. CD73 and Ly-6A/E expression therefore identify a population of Thpp-like cells in C57BL/6 mice and at least some other Ly-6.2 mice.
Maternal immunological tolerance of the semiallogeneic fetus involves several overlapping mechanisms to balance maternal immunity and fetal development. Anti-paternal CD8+ T cells are suppressed ...during pregnancy in some but not all mouse models. Since semen has been shown to mediate immune modulation, we tested whether exposure to paternal Ag during insemination activated or tolerized anti-paternal CD8+ T cells. The uterine lumen of mated female mice contained male MHC I+ cells that stimulated effector, but not naive, CD8+ T cells ex vivo. Maternal MHC class I+ myeloid cells fluxed into the uterine lumen in response to mating and cross-presented male H-Y Ag to effector, but not naive, CD8+ T cells ex vivo. However, neither unprimed nor previously primed TCR-transgenic CD8+ T cells specific for either paternal MHC I or H-Y Ag proliferated in vivo after mating. These T cells subsequently responded normally to i.p. challenge, implicating ignorance rather than anergy as the main reason for the lack of response. CD8+ T cells responded to either peptide Ag or male cells delivered intravaginally in ovariectomized mice, but this response was inhibited by systemic estradiol (inducing an estrus-like state). Subcutaneous Ag induced responses in both cases. Allogeneic dendritic cells did not induce responses intravaginally even in ovariectomized mice in the absence of estradiol. These results suggest that inhibition of antiallogeneic responses is restricted both locally to the reproductive tract and temporally to the estrous phase of the menstrual cycle, potentially decreasing the risk of maternal immunization against paternal Ags during insemination.
Human peripheral blood dendritic cells (PBDC) are a rare population comprised of several distinctive subsets. Analysis of these cells has been hindered by their low frequency. In this study, we ...report a novel direct
ex vivo 11-color flow cytometric assay that combines subset identification with analysis of activation status and endocytic ability of three major PBDC subsets (CD1c
+CD11c
+ “MDC1,” CD141
+CD11c
+ “MDC2,” and CD303
+CD11c
− “PDC”) within a single platform. This method eliminates the need for DC enrichment, isolation, or prolonged culture. Human peripheral blood mononuclear cells (PBMC) from healthy donors are incubated with FITC-dextran directly
ex vivo, prior to cell surface staining with various markers. As expected, PBDC identified by this assay express low levels of CD40 and CD86 directly
ex vivo, and significantly upregulate expression of these molecules upon stimulation with toll-like receptor ligands LPS and CpG oligonucleotides. In addition, PDC internalize FITC-labeled dextran poorly in comparison to MDC1 and MDC2 subsets. Specificity of FITC-dextran endocytosis is further verified by imaging flow cytometry. Furthermore, the combination of surface markers used in this assay reveals a previously unreported CD4
+CD11c
+CD303
−CD1c
−CD141
− cell population. Taken together, this assay is a rapid and cost-effective method that avoids manipulation of PBDC while providing direct
ex vivo high-dimensional flow cytometry data for PBDC studies.
Abstract
Intravenous (IV) anti-CD20 monoclonal antibody (mAb) therapy for chronic lymphocytic leukemia (CLL) patients often produces a first dose infusion reaction (FDIR) within the first 2 h that ...requires careful monitoring to avoid serious complications and fatalities. To better understand FDIR, we studied blood samples collected from 37 treatment-naïve CLL patients in our clinical trial NCT03788291 undergoing IV treatment with 50 mg of anti-CD20 mAb (rituximab). We analyzed four time points: baseline (prior to infusion), 1 h later (during infusion), at the end of infusion (~2.5 h) and at 48 h. Infusion reactions (CTCAE (v5) grade ≥2 events) were managed by interrupting the infusion and with symptomatic treatment. 24 (65%) patients had a FDIR and all patients completed the infusion.
Patient and CLL characteristics including measures of disease severity (Rai stage, IGHV mutation status, cytogenetic defects) did not correlate with FDIR. High levels of CLL cells and/or CD20 and their subsequent depletion resulting in decreased serum mAb and/or complement are thought to be risks for FDIR. However, measurements of these parameters did not correlate. Induction of cytokine release syndrome (CRS) may correlate with FDIR. However, CRS was observed in all samples after mAb infusion. The only significant associations with FDIR were higher levels of IP-10, IL-6, and IL-8, with IP-10 most significant. Anti-CD20 mAb activated cytotoxic effector cells, such as tissue-resident macrophages that ingest CLL cells via antibody-dependent cellular phagocytosis (ADCP), may be responsible for these events leading to FDIR. A mouse model of anti-CD20 mAb FDIR and in vitro ADCP cell systems will be useful to further understand the biology of FDIR.
Supported by funding from Acerta/AstraZeneca, the Cadregari Foundation, a generous donation by Elizabeth Aaron, and the NIH NCI grant number R21CA267040.
Respiratory syncytial virus (RSV) and influenza virus cause severe disease in elderly patients. The balance of pro- and anti-inflammatory cytokines may be critical in determining disease pathogenesis ...and outcome of infection. The frequencies of CD4 IL-10 (anti-inflammatory)- and CD4 and CD8 IFNγ (pro-inflammatory)-secreting memory T cells specific for either RSV or influenza were not significantly different between young and elderly groups, although the ratio of IL-10/IFNγ was significantly reduced in the elderly RSV response. A similar trend was seen for influenza. IFNγ-secreting CD4 T cells contributed significantly more to anti-RSV than anti-influenza responses in both groups.
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