scholarly journals Cryopreservation Decreases Receptor PD-1 and Ligand PD-L1 Coinhibitory Expression on Peripheral Blood Mononuclear Cell-Derived T Cells and Monocytes

2009 ◽  
Vol 16 (11) ◽  
pp. 1648-1653 ◽  
Author(s):  
D. E. Campbell ◽  
N. B. Tustin ◽  
E. Riedel ◽  
R. Tustin ◽  
J. Taylor ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Peripheral Blood ◽  
Peripheral Blood Mononuclear ◽  
Cell Receptors ◽  
Adult Control ◽  
Control Subjects ◽  
Tnf Α ◽  
Ifn Γ ◽  
The Impact

ABSTRACT The B7-CD28 immunoglobulin superfamily of costimulatory and coinhibitory ligands and their cell receptors play a critical role in modulating immune responses. Imbalances in these immune regulatory signals occur in pathological conditions characterized by chronic antigenic stimulation. Clinical studies often rely on the use of cryopreserved peripheral blood mononuclear cells (PBMC) to evaluate cellular immune responses. The impact of cryopreservation on these coinhibitory ligands and their cell receptors is unknown. In our studies, cryopreservation significantly reduced the expression of both PD-1 and PD-L1 on PBMC-derived CD3+/CD8+ T cells and CD45+/CD14+ monocytes obtained from adult control subjects. Blockade of PD-1, PD-L1, and PD-L2 using both freshly isolated and cryopreserved PBMC led to higher levels of phytohemagglutinin (PHA) and Candida-induced gamma interferon (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor alpha (TNF-α) with no effect on IL-10 production. Coinhibitory signaling blockade of freshly isolated, PHA-stimulated PBMC from normal adult controls and human immunodeficiency virus (HIV)-infected subjects led to increased production of IL-4 and IL-5. Candida-stimulated PBMC preferentially induced IFN-γ and TNF-α production, with reduced production of IL-2 and IL-10. This is in contrast to high levels of IFN-γ, IL-2, and TNF-α production with PHA-stimulated cells. The effects of coinhibitory blockade on PHA and Candida-induced lymphoproliferation were varied, with freshly isolated PBMC from adult control subjects and HIV-infected patients yielding higher levels of lymphoproliferation in response to PD-1/PD-L1 blockade. Immune function studies employing cryopreserved cells may lead to increased T-cell effector cytolytic and regulatory immune responses.

scholarly journals Th1/Th2 Cytokine Profile in Patients Coinfected with HIV and Leishmania in Brazil

2011 ◽  
Vol 18 (10) ◽  
pp. 1765-1769 ◽  
Author(s):  
Maria Zilma Andrade Rodrigues ◽  
Maria Fernanda Rios Grassi ◽  
Sanjay Mehta ◽  
Xing-Quan Zhang ◽  
Luana Leandro Gois ◽  
...  
Keyword(s):  
Immune Responses ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Content Type ◽  
Th2 Cytokine ◽  
Cytokine Levels ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

ABSTRACTTo evaluate the effects of HIV on immune responses in cutaneous leishmaniasis (CL), we quantified cytokine levels from plasma and stimulated peripheral blood mononuclear cells (PBMCs) from individuals infected with HIV and/or CL. Gamma interferon (IFN-γ) and interleukin 13 (IL-13) levels and the ratio of IFN-γ to IL-10 produced in response to stimulation with solubleLeishmaniaantigens were significantly lower in HIV-Leishmania-coinfected patients than in CL-monoinfected patients.

scholarly journals Multifunctional Human Immunodeficiency Virus (HIV) Gag-Specific CD8+ T-Cell Responses in Rectal Mucosa and Peripheral Blood Mononuclear Cells during Chronic HIV Type 1 Infection

2007 ◽  
Vol 81 (11) ◽  
pp. 5460-5471 ◽  
Author(s):  
J. William Critchfield ◽  
Donna Lemongello ◽  
Digna H. Walker ◽  
Juan C. Garcia ◽  
David M. Asmuth ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mononuclear Cells ◽  
Rectal Mucosa ◽  
Peripheral Blood Mononuclear ◽  
Tnf Α ◽  
Immunodeficiency Virus ◽  
Ifn Γ ◽  
Hiv 1

ABSTRACT The intestinal tract is a lymphocyte-rich site that undergoes severe depletion of memory CD4+ T cells within days of simian immunodeficiency virus or human immunodeficiency virus type 1 (HIV-1) infection. An ensuing influx of virus-specific CD8+ T cells, which persist throughout the chronic phase of infection, has also been documented in the gastrointestinal tract. However, little is known of the functionality of these effector cells or their relationship to the disease course. In this study, we measured CD8+ T-cell responses to HIV-1 peptides in paired rectal and blood samples from chronically infected patients. In both blood and rectum, there was an immunodominant CD8+ T-cell response to HIV Gag compared to Pol and Env (P < 0.01). In contrast, cytomegalovirus pp65 peptides elicited gamma interferon (IFN-γ) secretion strongly in peripheral blood mononuclear cells (PBMC) but weakly in rectal CD8+ T cells (P = 0.015). Upon stimulation with HIV peptides, CD8+ T cells from both sites were capable of mounting complex responses including degranulation (CD107 expression) and IFN-γ and tumor necrosis factor alpha (TNF-α) production. In rectal tissue, CD107 release was frequently coupled with production of IFN-γ or TNF-α. In patients not on antiretroviral therapy, the magnitude of Gag-specific responses, as a percentage of CD8+ T cells, was greater in the rectal mucosa than in PBMC (P = 0.054); however, the breakdown of responding cells into specific functional categories was similar in both sites. These findings demonstrate that rectal CD8+ T cells are capable of robust and varied HIV-1-specific responses and therefore likely play an active role in eliminating infected cells during chronic infection.

Donor DC Subsets Polarize Donor T-Cell Immune Responses in Allogeneic BMT.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 573-573
Author(s):  
Jian-Ming Li ◽  
Cynthia Giver ◽  
Doug McMillan ◽  
Wayne Harris ◽  
David L. Jaye ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Peripheral Blood ◽  
Immune Reconstitution ◽  
Hematopoietic Cell ◽  
Post Transplant ◽  
Donor T Cells ◽  
Ifn Γ

Abstract Introduction: Impaired or inappropriate immune reconstitution after allogeneic bone marrow transplantation (BMT) can lead to infection, graft-versus-host disease (GvHD) and leukemia relapse. We have previously reported that BM contains two populations of dendritic cell (DC) subsets, CD11b+ DC and CD11b− DC, and that CD11b depleted donor BM promoted increased donor T-cell chimerism and increased graft-versus-leukemia (GvL) activity in C57BL/6 → B10BR transplants [BBMT, 2004, 10: 540]. To explore the mechanism by which CD11b-depletion improved allo-reactivity, we performed allogeneic hematopoietic cell transplants using defined populations of donor stem cells, DCs, and T-cells in a MHC mis-matched BMT model. Methods: We transplanted FACS purified populations of 50,000 GFP+ CD11b- DC or CD11b+ DC in combination with 5,000 FACS purified Lin- Sca-1+ c-kit+ hematopoietic stem cells (HSC) and 300,000 or 1,000,000 congenic spleen T-cells from C57BL/6 donors into C57BL/6[H-2Kb], B10BR[H-2Kk] and PL/J[H-2Ku] recipients. Proliferation of CFSE stained donor T-cells was measured at 72 hours post-transplant. FACS cytometric bead array and intracellular cytokine staining measured serum and intracellular cytokines in donor T-cells. Results: The initial proliferation and Ki-67 expression of CFSE labeled donor T-cells in allogeneic recipients were much higher than in syngeneic recipients (homeostatic proliferation). Confocal microscopy showed co-localization of donor DC subsets with donor T-cells in the recipient spleens at 3 and 10 days post-transplant. In the allogeneic transplant settings, donor T-cells co-transplanted with CD11b- DC showed increased IFN-γ synthesis at 3 and 10 days post-transplant compared to donor T-cells co-transplanted with HSC plus CD11b+ DC or HSC alone. Increased proliferation of donor T-cells led to increased donor T-cell chimerism at day 10, 30, 60, and day105 post-transplant among recipients of CD11b- DC compared to recipients of HSC alone or HSC plus CD11b+ DC (Figure 1). Transplantation of spleen T-cells and CD11b- DC did not increase GvHD, but was associated with full donor chimerism. In contrast, transplantation of allogeneic CD11b+ DC led to persistence and expansion of residual host T-cells (Figure 2), increased numbers of donor CD4+CD25++Foxp3+ T-cells, and higher serum level of IL-10 supporting early post-transplant expansion of donor T regulatory cells (Treg). Conclusions: Donor CD11b- DC promoted immune reconstitution by polarizing donor T-cells to Th1 immune responses associated with increased IFN-γ synthesis and donor T-cell proliferation, while donor CD11b+ DC suppressed immune reconstitution by inhibiting donor T-cell allogeneic immune responses. These data support a novel paradigm for the regulation of post-transplant immunity and suggest clinical methods to test the hypothesis that manipulation of the DC content of a hematopoietic cell allograft regulates post transplant immunity in the clinical setting. Figure 1. Donor Spleen Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(+)DC and spleen T-cells] Figure 1. Donor Spleen Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(+)DC and spleen T-cells] Figure 2. Host Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(-)DC and spleen T-cells] Figure 2. Host Derived T-cells in Peripheral Blood [* p<0.05, v.s. recipients of HSC plus CD11b(-)DC and spleen T-cells]

Preliminary biomarker and pharmacodynamic data from a phase I study of single-agent bispecific antibody T-cell engager GBR 1302 in subjects with HER2-positive cancers.

2018 ◽  
Vol 36 (5_suppl) ◽  
pp. 69-69 ◽  
Author(s):  
Martin Wermke ◽  
Juergen Alt ◽  
John S. Kauh ◽  
Jonathan Back ◽  
Yacine Salhi ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Solid Tumors ◽  
Peripheral Blood ◽  
Bispecific Antibody ◽  
Cell Populations ◽  
Single Subject ◽  
Her2 Positive ◽  
Tnf Α ◽  
Ifn Γ

69 Background: HER2 is overexpressed in many solid tumors and is a validated therapeutic target. GBR 1302 is a HER2xCD3 bispecific antibody engineered (using Glenmark’s BEAT® platform) to direct T-cells to HER2-expressing tumor cells. GBR1302-101 (NCT02829372) is an ongoing, multicenter, open-label, first-in-human study of GBR 1302 in subjects with HER2-positive cancers to evaluate the safety, tolerability, and preliminary efficacy of GBR 1302, and to elucidate the mechanism(s) by which it redirects T-cells to tumor and enhances cytolytic activity of cytotoxic T-cells. Methods: Adults with progressive HER2-positive solid tumors with no available standard or curative treatment receive intravenous GBR 1302 on Day 1 and Day 15 in 28-day treatment cycles at escalating dose levels, starting at 1 ng/kg. The first 4 cohorts consist of a single subject; subsequent cohorts enroll using a 3+3 design. The primary and secondary efficacy and safety endpoints of this trial will be reported at the end of the study. Preliminary pharmacodynamic (PD) data are reported for cellular biomarkers and cytokines as assessed by FACS and ELISA in peripheral blood. Results: Beginning at 30 ng/kg dosing of GBR 1302 (Cohort 4), numbers of peripheral blood CD3, CD4, and CD8 positive T-cell populations decreased within 6 hours of initiating administration, but recovered to levels at or above baseline by 48 hours. A parallel, transient increase was observed in peripheral blood cytokines (IL-2, IL-6, IL-10, IFN-γ, TNF-α). At doses greater than 30 ng/kg, more pronounced cytokine increases were observed, which normalized at 12 hours. At the highest dose level for which data are available (n = 8 subjects; Cohort 5), changes from baseline in cytokine expression at ~340 hours were greater by ~60-fold for IL-6, ~30-fold for IL-2, ~3-fold for IFN-γ, ~5-fold for TNF-α, and ~18-fold for IL-10. Two subjects treated at 100 ng/kg experienced Grade 1 cytokine release syndrome, evidenced by short-lived fever spikes. Dose escalation is ongoing. Conclusions: Preliminary PD data indicate changes in peripheral T-cell populations and inflammatory cytokines following GBR 1302 treatment. Clinical trial information: NCT02829372.

scholarly journals Comparative analysis of cellular immune responses in conventional and SPF Baboons (Papio spp.)

2018 ◽  
Author(s):  
Elizabeth R Magden ◽  
Bharti P. Nehete ◽  
Sriram Chitta ◽  
Lawrence E. Williams ◽  
Joe H Simmons ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Lymphocyte Subsets ◽  
Effector Memory ◽  
Peripheral Blood Mononuclear ◽  
Phenotypic Differences ◽  
Successful Model ◽  
Specific Pathogen ◽  
Ifn Γ ◽  
Cellular Immune

AbstractBaboons (papio spp.) have served as a successful model of human disease such as cardiac and respiratory, infectious, diabetes, genetics, immunology, aging, and xenotransplantation. The development of an immunologically defined specific-pathogen free (SPF) baboon model has further advanced research, especially with studies involving the immune system and immunosuppression. In this study, we compare normal immunological changes of peripheral blood mononuclear cell (PBMC) subsets, and their function in age-matched conventional and SPF baboons. Our results demonstrate that both groups have comparable numbers of different lymphocyte subsets, but there are phenotypic differences in central and effector memory T cells subsets that are more pronounced in the CD4+ T cells. Despite equal proportions of CD3+ T cells among the conventional and SPF baboon groups, PBMC show higher proliferative responses to mitogens PHA and PWM and higher IFN-γ producing cells to Con A and PWM in the conventional group. Plasma levels of the inflammatory cytokine TNF-α were significantly higher in SPF baboons. Exposure of PBMC from conventional baboons to various Toll like ligands (TLR ligands) TLR-3, TLR-4 and TLR-8 show higher IFN-γ producing cells while PBMC from SPF baboons stimulated with TLR-5 and TLR-6 ligand show higher IFN-γ producing cells. These findings suggest that while the lymphocyte subsets in conventional and SPF baboons share many phenotypic and functional similarities, specific differences exist in immune function of lymphocytes which could impact the quality and quantity of innate and adaptive immune responses. These differences should be considered for better experimental outcomes, specifically in studies measuring immunological endpoints.

CD8+CD28− T cells: key cytotoxic players impacting disease pathogenesis in chronic HBV infection

2019 ◽  
Vol 133 (17) ◽  
pp. 1917-1934
Author(s):  
Madhuparna Nandi ◽  
Sourina Pal ◽  
Sumantra Ghosh ◽  
Bidhan Chandra Chakraborty ◽  
Debangana Dey ◽  
...  
Keyword(s):  
T Cells ◽  
Chronic Hepatitis ◽  
T Cell ◽  
Hepatitis B ◽  
Chronic Hepatitis B ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Hepatitis B E Antigen ◽  
Tnf Α ◽  
Ifn Γ

Abstract During chronic hepatitis B (CHB), CD8+ T cells down-regulate CD28, the primary co-stimulation molecule for T-cell activation. Diverse functional attributes of CD8+CD28− T cells are suggested in various disease contexts. The present study aimed to characterize CD8+CD28− T cells in different phases of chronic Hepatitis B virus (HBV) infection (CHI)- Immune-tolerance (IT), Hepatitis B e-antigen-positive CHB (EP-CHB), Inactive carriers (IC) and Hepatitis B e-antigen-negative CHB (EN-CHB), to appraise their contribution in HBV-related disease pathophysiology. Flow cytometry analysis of T cells in peripheral blood of study subjects revealed enhanced CD8+CD28− T-cell accumulation in EP-/EN-CHB, compared with IT/IC and they expanded equivalently in HBV-specific and non-specific CD8+ T-cell compartments. Profound increase in CD8+CD28− T cells expressing perforin/granzyme-B/CD57/IFN-γ/TNF-α and markers of terminal differentiation were observed exclusively in EP-/EN-CHB. Further, activation with anti-NKG2D resulted in heightened IFN-γ/TNF-α production selectively from CD8+CD28− T cells, suggesting NKG2D-mediated alternative co-stimulation. CD8+CD28− T cells sorted from CHB patients induced enhanced apoptosis of peripheral blood mononuclear cells (PBMC), including CD4+ T cells. However, NKG2D-ligand (major histocompatibility complex class I chain-related molecule A/B (MICA/B)) was preferentially expressed by HBV-specific CD4+ T cells of CHB patients, making these cells a potential target to NKG2D-dependent CD8+CD28− T-cell killing. Both CD28+ and CD28− T cells in CHB expressed CXCR3 at similar levels and thus capable of homing to the liver. A positive correlation was seen between CD8+CD28− T-cell frequency and serum-alanine transaminase (ALT) levels and CHB-derived CD8+CD28− T cells caused pronounced cell death in HBV-transfected Huh7 cells. Immunofluorescence staining identified greater intrahepatic incidence of CD8+CD28− T cells but decline in CD4+ T cells in CHB than IC. Collectively, CD8+CD28− T cells demonstrated differential distribution and phenotypic/functional skewing in different CHI phases and contribute to disease progression by Perforin-Granzyme- or IFN-γ-TNF-α-mediated cytotoxicity while restraining antiviral immunity through NKG2D-dependent HBV-specific CD4+ T-cell depletion.

PBMC-06- Intracellular Cytokine Staining (ICS) of IFN-gamma, IL-4 and IL-17 on Human Peripheral Blood Mononuclear Cells (PBMC) v1

2021 ◽  
Author(s):  
Alessia Furgiuele ◽  
Emanuela Rasini ◽  
Maria Giulia Albizzati ◽  
Alessandra Luini ◽  
Marco Ferrari ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Brefeldin A ◽  
Intracellular Cytokine ◽  
Peripheral Blood Mononuclear ◽  
Blood Mononuclear Cells ◽  
Ifn Γ

This present protocol is developed to analyze the frequency of IFN-γ-, IL-4- and IL-17-producing CD4+T cells, identified from ex vivo human peripheral blood mononuclear cells (PBMC). The frequencies of cytokine producing cells derived from activation of PBMC was induced trough the stimulus phorbol 12-myristate 13-acetate (PMA) and ionomycin. According onpreviously published protocols concentrations of stimulating substances were in the range from 10, to 50 ng/ml for PMA and 1 µg/ml for ionomycin (Gupta and Maecker, 2015; Foster et al., 2007; Freer and Rindi, 2013; https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). The PMA concentrations of 10, 20 and 50 ng/ml were tested and finally the PMA concentration of 10 ng/ml was chosen since it was sufficient to obtain a frequency of cytokines comparable to that obtained with higher stimulus concentrations. PMA/ionomycin and brefeldin A are incubate together for a time of 5 h (Gupta and Maecker, 2015, Foster et al., 2007, Freer and Rindi, 2013, https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). The protein secretion inhibitor brefeldin A, was used at the concentration of 10 µg/ml (Gupta and Maecker, 2015; Foster et al., 2007; Freer and Rindi, 2013). Cell concentrations may vary in a range from 2.5 x106 to 10 x106 cells/ml (Maecker, 2004; Freer and Rindi, 2013a; https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.in.560751.pdf). Concentration of 1x106 cells/ml, 4x106 cells/ml and 8x106cells/ml were tested. Cell tritation have shown a higher functional response proportional to the cell concentration when exposed to a fixed concentration of stimulants. Cell concentration of 8 milions/ml was selected in order to obtain the higher percentage of IFN-γ-, IL-4- and IL-17-producing CD4+T cells. In conclusion the present protocol provides that, for a optimal optimal percentage of IFN-γ-, IL-4- and IL-17-producing CD4+T cells as assessed by flow cytometry (Table 1), PBMC in a concentration 8 milions/ml were stimulated with PMA 10 ng/ml and ionomycin 1 µg/ml, and cultured for 5 h in presence of brefeldin A 10 µg/ml according to the procedure described in detail below. References Baran, J., Kowalczyk, D., Ozog, M., Zembala, M., 2001. Three-color flow cytometry detection of intracellular cytokines in peripheral blood mononuclear cells: Comparative analysis of phorbol myristate acetate-ionomycin and phytohemagglutinin stimulation. Clin. Diagn. Lab. Immunol. 8, 303–313. https://doi.org/10.1128/CDLI.8.2.303-313.2001 Foster, B., Prussin, C., Liu, F., Whitmire, J.K., Whitton, J.L., 2007. Detection of intracellular cytokines by flow cytometry. Curr. Protoc. Immunol. Chapter 6. https://doi.org/10.1002/0471142735.im0624s78 Freer, G., Rindi, L., 2013. Intracellular cytokine detection by fluorescence-activated flow cytometry: Basic principles and recent advances. Methods 61, 30–38. https://doi.org/10.1016/j.ymeth.2013.03.035 Gupta, S., Maecker, H., 2015. Intracellular Cytokine Staining (ICS) on Human Lymphocytes or Peripheral Blood Mononuclear Cells (PBMCs). BIO-PROTOCOL 5. https://doi.org/10.21769/bioprotoc.1442 Maecker, H.T., 2004. Cytokine flow cytometry. Methods Mol. Biol. 263, 95–108. https://doi.org/10.1385/1-59259-773-4:095 https://www.bdbiosciences.com/content/bdb/paths/generate-tds-document.us.560751.pdf BEFORE STARTING with this procedure Moreover, work under laminar flow hood when you are processing samples from the beginning to the end of the culture. Make sure you are using, sterile culture medium and sterile plastic disposable as well.

Anti-Leukemia Immune Responses in CML Patients Treated with Imatinib Mesylate.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1108-1108
Author(s):  
Christiane I.-U. Chen ◽  
Holden T. Maecker ◽  
Wesley H. Neal ◽  
Rhoda Falkow ◽  
Peter P. Lee
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Immune Responses ◽  
Cd4 T Cells ◽  
Post Treatment ◽  
Leukemia Cells ◽  
Tnf Α ◽  
Ifn Γ ◽  
Cytogenetic Remission ◽  
Pre Treatment

Abstract Imatinib mesylate, a selective inhibitor of the bcr/abl tyrosine kinase, has revolutionized the treatment of patients with chronic myelogenous leukemia (CML). Most CML patients in chronic phase achieve hematologic remission with imatinib, while some achieve cytogenetic remission. As imatinib is an oral agent with few side effects, it has rapidly become the first-line therapy for most CML patients. However, this therapy does not represent a cure, as patients who discontinue the drug invariably relapse. Furthermore, imatinib resistance is beginning to emerge in some patients. Hence, the need to find alternate, potentially curative, therapies for CML remains. To date, the only curative treatment for CML is allogeneic bone marrow or stem cell transplantation (ABMT). A major mechanism of the curative potential of ABMT is immunological, as evidenced by the poor clinical outcome with T cell-depleted ABMT, and the efficacy of donor lymphocyte infusions (DLI) upon relapse. We hypothesized that an effective anti-leukemia immune response may emerge in patients entering remission on imatinib which may contribute to its clinical effectiveness. If so, strategies to further enhance this anti-leukemia immune response may lead to a potential cure. To determine if CML patients in remission on imatinib develop anti-leukemia immune responses, blood and bone marrow samples from patients before and after treatment were collected and analyzed. Pre-treatment samples were utilized as sources of autologous leukemic cells to detect anti-leukemia immune responses in post-treatment samples in IFN-g ELISPOT assays. Pre-treatment samples alone, post-treatment samples alone, and when available, serial post-treatment samples mixed together served as controls. In 9 of 14 patients investigated, IFN-g release was detected in pre- and post-treatment samples together with a median response of 22 spots above background (range 10 – 56 dots, p&lt;0.01), whereas serial post-treatment samples together in 8 patients yielded results similar to background (median 5, range 5 – 20). In 6 of these patients in hematologic (or cytogenetic) remission, sufficient cells were available to allow additional analyses via intracellular staining for IFN-g, TNF-a, and IL-2 in autologous leukemia stimulated T cells (CD4 and CD8) and NK cells. In 4 of 6 patients, leukemia-reactive T cells were detected, most prominently in CD4+ T cells expressing TNF-a (1.4 – 37%), followed by IL-2 (0.3 – 12%) and IFN-g (0.1 – 4.6%). NK cells did not show significant expression of these cytokines upon stimulation with autologous leukemia cells. In pre-treatment and post-treatment samples alone, IL-2, TNF-a, and IFN-g expression was not detectable (0 – 0.5%). These results suggest that a significant portion of CML patients in remission with imatinib develop an anti-leukemia immune response, most notably in CD4+ T cells. Mechanisms by which imatinib treatment leads to anti-leukemia immune responses, and the molecular targets to which these cells are directed, will be further investigated. This knowledge will be useful in the development of immunotherapy strategies against CML as well as other leukemias, and raises the hope that immunotherapy may be combined with imatinib to eradicate residual leukemia cells for a durable cure of the disease. intracellular cytokine staining CD4+ T Cells CD8+ T Cells IL-2 IFN- γ TNF- α IL-2 IFN- γ TNF- α pt 1 0.3 0 0.8 0.1 0.1 0.5 pt 1 0.3 0.1 1.4 0.1 0.1 0.4 pt 2 2.6 0.8 10.3 2.2 2.1 6.1 pt 3 21 2 37 2.3 0.7 1.7 pt 4 12 4.6 19 6.3 1.8 5.8

Boost and Loss Of Immune Responses Against Tumor-Associated Antigens In The Course Of Pregnancy As a Model For Immunotherapy

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4505-4505 ◽  
Author(s):  
Mathias Lutz ◽  
Medical Student ◽  
Miriam Alb ◽  
Andrea Worschech ◽  
Sabine Gahn ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Prospective Study ◽  
Pregnant Women ◽  
Longitudinal Analysis ◽  
Peripheral Blood ◽  
Delivery Time ◽  
Cross Sectional ◽  
Ifn Γ ◽  
Time Point

Background The therapeutic potential of allogeneic hematopoietic stem cell transplantation (HSCT) mainly relies on graft-versus-leukemia effects mediated by donor-derived T cells targeting both minor histocompatibility antigens and tumor-associated antigens (TAAs). The efficacy of TAA-mediated immune responses particularly depends on the level of antigen expression in the target tissue and the presence of a proinflammatory immune environment allowing donor-derived T cells to overcome peripheral tolerance and to attack the malignant cells in a selective manner. As several TAAs with clinical relevance in tumor and/or transplant immunotherapy such as Mucin 1 (MUC-1), Preferentially expressed antigen of melanoma (PRAME), Wilms tumor protein 1 (WT1) and Human epidermal growth factor receptor 2 (HER2/neu) are highly expressed in the placental tissue we investigated the effects of pregnancy on the frequency of immune responses against these TAAs in a prospective study. A cross-sectional analysis in a previously reported group of 114 healthy volunteer blood donors revealed no significant effects of prior pregnancies. Here, we present the results of the longitudinal analysis in women during and after their first pregnancy. Materials and Methods After local ethical approval and written informed consent a total of 44 HLA-A*02:01-positive women during their first pregnancy were enrolled in this prospective study. Immune responses against TAAs were assessed at four different time points: First or second trimester (Time point A, n=43), 1 to 3 days after delivery (Time point B, n=31), 6 to 8 weeks after delivery (Time point C, n=34) and after completed nursing (Time point D, n=14). At each time point peripheral blood was drawn and peripheral blood mononuclear cells (PBMCs) were collected by density gradient centrifugation. Isolated CD8+ T cells were stimulated with irradiated T2 cells which were loaded with immunodominant peptides (0.1 and 10 µM) of HER2/neu, MUC-1, PRAME, and WT1. Interferon-gamma (IFN-γ) mRNA expression was measured by quantitative polymerase chain reaction (qPCR) after extraction of total RNA. IFN-γ mRNA expression was calculated as relative fold change compared to the irrelevant melanoma antigen Glycoprotein 100 (gp100). A two-fold change was considered as cut-off for positive results. A HLA-A*02:01 restricted CMV peptide was used as positive control. Results All results are presented in Figure 1. Positive immune responses are presented above the cut-off line. A significant number of positive immune responses against MUC-1, PRAME and WT1 were found during pregnancy (Group A) and early after delivery (Group B). The frequency of positive immune responses against these TAAs decreased during and/or after the period of nursing leaving only one borderline significant finding behind (Group C and D). Against HER2/neu there were none or only single events of immune responses detectable in any group. Conclusions The results from this longitudinal analysis show that immune responses against the transplant-relevant TAAs MUC-1, PRAME and WT1 are detectable in pregnant women but are lost after delivery. These findings are in line with our observations from the inter-individual cross-sectional study where women with prior pregnancy did not have a significantly increased frequency of immune responses against TAAs compared to women without prior pregnancies. The abrogation of the immune response as shown in a longitudinal manner is likely to be a result of an immunosuppressive environment induced by increasing levels of steroid hormones during pregnancy. As immune responses against TAAs can be found also in non-pregnant women we assume that the overexpression of TAAs in the placenta induces a boost in autoimmune responses that is consequently compromised by the immunosuppressive environment. Therefore pregnancy may be a useful model for the boost and the regulation of immune responses against TAAs with implications for immunotherapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Host Immune Responses to Chronic Adenovirus Infections in Human and Nonhuman Primates

2008 ◽  
Vol 83 (6) ◽  
pp. 2623-2631 ◽  
Author(s):  
Roberto Calcedo ◽  
Luk H. Vandenberghe ◽  
Soumitra Roy ◽  
Suryanarayan Somanathan ◽  
Lili Wang ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Peripheral Blood ◽  
Neutralizing Antibodies ◽  
Potential Role ◽  
Low Frequencies ◽  
Host Immune Responses ◽  
Virus Genomes ◽  
The Impact

ABSTRACT Recent studies indicate that great apes and macaques chronically shed adenoviruses in the stool. Shedding of adenovirus in the stool of humans is less prevalent, although virus genomes persist in gut-associated lymphoid tissue in the majority of individual samples. Chimpanzees have high levels of broadly reactive neutralizing antibodies to adenoviruses in serum, with very low frequencies of adenovirus-specific T cells in peripheral blood. A similar situation exists in macaques; sampling of guts from macaques demonstrated adenovirus-specific T cells in lamina propria. Humans show intermediate levels of serum neutralizing antibodies, with adenovirus-specific T cells in peripheral blood of all individuals sampled and about 20% of samples from the gut, suggesting a potential role of T cells in better controlling virus replication in the gut. The overall structure of the E3 locus, which is involved in modulating the host's response to infection, is degenerate in humans compared to that in apes, which may contribute to diminished evasion of host immunity. The impact of adenovirus persistence and immune responses should be considered when using adenoviral vectors in gene therapy and genetic vaccines.

Sign in / Sign up

Export Citation Format

Share Document