Treg depletion with denileukin difititox (DD) enhances lymphocytosis and eosinphilia in patients treated with high-dose IL-2 (HDIL-2) for metastatic renal cell cancer (MRCC)

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 14627-14627
Author(s):  
A. Gidron ◽  
J. Eklund ◽  
B. Mortone ◽  
A. W. Rademaker ◽  
C. Goolsby ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
T Cells ◽  
Cell Cancer ◽  
Peak Level ◽  
Absolute Number ◽  
High Dose ◽  
Immune Effector ◽  
Treg Depletion ◽  
Group A

14627 Background: CD4+CD25+ T cells (Treg) play a suppressive role in immune regulation. DD is an IL-2 receptor specific cytotoxin. We postulated depletion of Treg with DD may enhance immune effector cell populations during HDIL-2 tx, including eosinophilia which was reported to be involved in immune response to neoplasm (Mattes et al. J Exp Med 197: 387, 2003). Methods: Seven pts (5 male, median age 58 yrs) with MRCC were tx’d with HDIL-2 and DD in different schedules to determine safety and effect on immune response as manifested by changes in Treg, lymphocyte, and peak eosinophil counts. Pts were tx’d with IL-2 600,000 IU/kg Q8H on days (d) 1–5 and 15–19. Three (group A) and 2 (group B) pts were given 6 and 9 ug/kg daily on d 8–10 respectively, while 2 (group C) pts received 9 ug/kg of DD on d -4- -2. Four (group D) pts with metastatic melanoma who received HDIL-2 as above but without DD were included as controls. Flow cytometry was done on days -4, 1,8,10,15 for group C and on days 1, 8, 10, 15, 22 for groups A, B, and D. CBC was obtained within 24 hours of flow cytometry. Results: After DD Treg increased in group A (mean change in absolute T-reg count of 16%) and decreased in groups B and C (34.5 and 20% respectively) compared to baseline. Group C trended toward a greater lymphocytosis at day 8 compared to all other groups (mean increase of 8.6 vs. 3.3 K/ul p = 0.059). A higher peak level of eosinophilia was noted in group C compared with groups A, B and D combined (mean increase of 9.9 vs. 3.0 k/ul p = 0.03). Group C demonstrated a higher mean % change in absolute number of CD8+ T-cells between onset of therapy and Day 8 compared to groups A, B, D combined (increase of 1095% vs. 496% respectively, p = 0.35). Toxicity was similar to that expected with HDIL-2. Conclusions: Administration of DD in conjunction with HDIL-2 was associated with a decrease in Treg that may be schedule and dose dependent. The results suggest an enhanced immune stimulatory effect as manifested by lymphocytosis and peak eosinophilia and CD8+ T-cells in group C. Despite small pt numbers, results suggest that pre-treatment with DD may confer an advantage. It is too early to know if laboratory results correlate to clinical benefit. [Table: see text]

Concurrent Treatment with Denileukin Diftitox (DD) To Deplete T-Regulatory Cells Enhances Rebound Lymphocytosis and Eosinophilia in Patients Treated with High-Dose IL-2 (HDIL-2) for Metastatic Renal Cell Cancer (MRCC).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1729-1729
Author(s):  
Adi Gidron ◽  
John Eklund ◽  
Brenda Martone ◽  
Alfred W. Rademaker ◽  
Charles Goolsby ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
Statistical Significance ◽  
Cell Cancer ◽  
Peak Level ◽  
High Dose ◽  
Maximal Increase ◽  
Group A ◽  
Group B ◽  
Group D

Abstract Background: CD4+CD25+hi T cells (Treg) play a suppressive role in immune regulation. DD is an IL-2 receptor specific cytotoxin. We postulated depletion of Treg with DD may enhance immune effector cell populations after HDIL-2 treatment, including rebound lymphocytosis and also eosinophilia which has been reported to be involved in immune response to neoplasm (Mattes J Exp Med 197: 387, 2003). Methods: In this pilot study, 12 pts (8 male, median age 58 yrs) with MRCC were tx with HDIL-2 and DD in different schedules to determine safety and effect on immune response as manifested by changes in Treg, peak lymphocyte, and eosinophil counts. Pts were treated with IL-2 600,000 IU/kg Q8H on days (d) 1–5 and 15–19. Three (group A) and 4 (group B) pts were given 6 and 9ug/kg daily on d8–10 respectively, while 5 (group C) pts received 9ug/kg of DD on d −4 to −2. Nine (group D) pts with metastatic melanoma who received HDIL-2 as above but without DD were included as controls. Flow cytometry was done on days −4, 1,8,10,15,22 for group C and on days 1,8,10,15,22 for groups B, and D. CBC was obtained concurrent or within 24 hours of flow cytometry. Group A pts were evaluated for safety only and were excluded from analysis. Results: Prior to enrollment, all pts had undergone nephrectomy and four patients received interferon-alpha. One pt from group B withdrew from study and was not included in analysis. Administration of DD resulted in a median decline of 25% in Treg number (not significant). DD given before HDIL-2 was associated with a greater increase in Treg post HDIL-2. In Group C there was an increase of rebound median Treg count of 0.88k/ul compared with 0.060k/ul in group B (p=0.025). Absolute lymphocytosis was higher in the combined group getting DD compared to control (median maximal increase of 7.6 vs 4.7 k/ul, respectively) although the difference did not reach statistical significance. However, group C pts had a greater increase in absolute lymphocytosis than did group B pts in which absolute lymphocytosis actually decreased (median increase 10.6 vs. median decrease 0.4 k/ul, p=0.025). A higher peak level of eosinophilia was noted in groups B and C compared with group D (mean increase of 10.5 vs. 4.0 k/ul p=0.2). Group C had a greater peak eosinophilia than group B (11.2 vs 2.2 k/ul p=0.053) Toxicity was manageable and consistent with those seen with HDIL-2. Median HDIL-2 dose given was 21 (range, 14–28). No clinical responses were observed. Of 11 pts included in the analysis 1 pt from group A expired 68 weeks after enrollment. All remaining patients are alive. Survival from enrollment ranges from 11 to 93 weeks. Conclusion: Overall, the combination of DD and HDIL-2 results in a stimulatory effect as manifested by increased rebound lymphocytosis and eosinophilia compared to HDIL-2 alone. Administration of DD in conjunction with HDIL-2 was associated with a rebound in Treg that may be schedule and dose dependent. The results suggest an enhanced immune stimulatory effect as manifested by lymphocytosis and peak eosinophilia in group C. However, this stimulatory effect also extends to Treg that may prove detrimental clinically. Further exploration of these effects in immunotherapy naïve patients would be beneficial.

scholarly journals Netrin-1 reduces lung ischemia-reperfusion injury by increasing the proportion of regulatory T cells

2020 ◽  
Vol 48 (6) ◽  
pp. 030006052092641
Author(s):  
Zhili Chen ◽  
Yuxi Chen ◽  
Jue Zhou ◽  
Yong Li ◽  
Changyao Gong ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Regulatory T Cells ◽  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Axonal Guidance ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Treg Depletion

Objective Inflammation is the primary mechanism of lung ischemia-reperfusion injury (LIRI) and neurologic factors can regulate inflammatory immune responses. Netrin-1 is an axonal guidance molecule, but whether Netrin-1 plays a role in LIRI remains unclear. Methods A mouse model of LIRI was established. Immunohistochemistry was used to detect expression of Netrin-1 and to enumerate macrophages and T cells in lung tissue. The proportion of regulatory T cells (Tregs) was assessed by flow cytometry. Levels of apoptosis were assessed by terminal deoxynucleotidyl transferase dUTP nick end staining. Results Numbers of macrophages and T cells in the lung tissues of mice with LIRI were elevated, while expression of netrin-1 was significantly decreased. Flow cytometry showed that the proportion of Tregs in mice with LIRI was significantly decreased. The proportion of Tregs among lymphocytes was positively correlated with netrin-1 expression. In vitro experiments showed that netrin-1 promoted an increase in Treg proportion through the A2b receptor. Animal experiments showed that netrin-1 could inhibit apoptosis and reduce T cell and macrophage infiltration by increasing the proportion of Tregs, ultimately reducing LIRI. Treg depletion using an anti-CD25 monoclonal antibody blocked the effects of netrin-1. Conclusion Netrin-1 reduced LIRI by increasing the proportion of Tregs.

The Absence of Vasoactive Intestinal Peptide Augments Allo-Reactivity and the Anti-Viral Response in Bone Marrow Transplantation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3267-3267
Author(s):  
Lauren T. Southerland ◽  
Jian-Ming Li ◽  
Sohrab Hossain ◽  
Cynthia Giver ◽  
Wayne Harris ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
Cell Response ◽  
Low Dose ◽  
T Cell Response ◽  
High Dose ◽  
Mcmv Infection

Abstract Background: The severe morbidity and mortality associated with bone marrow transplantation (BMT) is caused by uninhibited immune responses to alloantigen and suppressed immune responses to pathogens. Vasoactive Intestinal Peptide (VIP) is an immunomodulatory neuropeptide produced by T-cells and nerve fibers in peripheral lymphoid organs that suppresses immune responses by induction of tolerogenic dendritic cells. In order to determine the immunoregulatory effects of VIP, we examined T-cell immune responses to allo- and viral-antigens in VIP knockout (KO) mice and mouse BMT recipients of hematopoietic cells from VIP KO donors. Methods: VIP KO mice and VIP WT littermates were infected with lethal or sub-lethal doses (5 × 104− 5 × 105 PFU) of murine cytomegalovirus (mCMV) and the T-cell response to viral antigen was measured by flow cytometry for mCMV peptide-MHC class 1-tetramer+ CD8+ T-cells. We transplanted 5 × 106 BM plus 1 × 106 splenocytes (SP) either from VIP KO or VIP WT donors in an C57BL/6 to F1(BL/6 × Balb/c) allo-BMT model and assessed survival, GvHD, donor T-cell expansion, chimerism, and response to mCMV vaccination and mCMV infection. Results: B-cell, αβ and γδ T-cell, CD8+ T-cell, CD11b+ myeloid cell, and dendritic cell numbers were equivalent between VIP KO and WT mice, while VIP KO mice had higher number of CD4+ and CD4+CD62L+CD25+ T-cells. Non-transplanted VIP KO mice survived mCMV infection better compared to VIP WT, with a brisker anti-viral T-cell response in the blood. In the allogeneic BMT setting, recipients of VIP KO BM plus VIP KO SP had more weight loss and lower (40%) 100 day post-transplant survival compared to the recipients of VIP KO BM plus WT SP (80% survival), recipients of WT BM plus KO SP (100% survival), and recipients of WT BM plus WT SP (80% survival). Recipients of VIP KO grafts had a significantly greater anti-mCMV response that peaked four days earlier than the tetramer response of mice transplanted with WT cells. This increased anti-viral response to vaccination correlated with a greater and more rapid T-cell response to secondary viral challenge. Conclusions: These experiments suggest that the absence of all VIP in the body, or the absence of VIP in a transplanted immune system, enhances anti-viral immunity and allo-immune responses. Modulation of the VIP pathway is a novel method to regulate post-transplant immunity. Figure 1: VIP knockout(KO) mice have an increased CMV tetramer response. VIP KO and VIP WT mice were infected (day 0) with either a sub-lethal low dose (5 × 10^4 PFU) or a lethal high dose (5 × 10^5 PFU) of CMV. Peripheral blood was stained for T cell markers and tetramer and analyzed by flow cytometry. On day 3, high dosed VIP KO mice had a higher number of tetramer positive CD8 T cells and better survival than WT mice (all high dose VIP WT died prior to day 10). VIP KO mice had a significant increase in tetramer positive CD8 T cells between days 3 and 10. *** p<0.01, difference between VIP KO and VIP WT littermate at designated dose level and day. Figure 1:. VIP knockout(KO) mice have an increased CMV tetramer response. VIP KO and VIP WT mice were infected (day 0) with either a sub-lethal low dose (5 × 10^4 PFU) or a lethal high dose (5 × 10^5 PFU) of CMV. Peripheral blood was stained for T cell markers and tetramer and analyzed by flow cytometry. On day 3, high dosed VIP KO mice had a higher number of tetramer positive CD8 T cells and better survival than WT mice (all high dose VIP WT died prior to day 10). VIP KO mice had a significant increase in tetramer positive CD8 T cells between days 3 and 10. *** p<0.01, difference between VIP KO and VIP WT littermate at designated dose level and day.

High-Dose RHAMM-R3 Peptide Peptide Vaccination for Patients with Vaccination for Patients with Acute Myeloid Leukemia (AML), Myelodysplastic Syndrome (MDS), Multiple Myeloma (MM) and Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2911-2911 ◽  
Author(s):  
Jochen Greiner ◽  
Anita Schmitt ◽  
Krzysztof Giannopoulos ◽  
Isabel Funk ◽  
Marta Heyduk ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
T Cells ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
High Dose ◽  
Clinical Effects ◽  
Peptide Vaccination

Abstract We have demonstrated immunological responses and positive clinical effects of a peptide vaccination for patients with AML, MDS, MM and CLL with a limited tumor load or a minimal residual disease over-expressing RHAMM using 300 μg RHAMM-R3 peptide (Schmitt et al., Blood 2008; Giannopoulos et al., abstract submitted). To date, 26 patients were enrolled in this clinical peptide vaccination trial. Here, we report on the second cohort of nine patients with AML, MDS and MM vaccinated with a higher peptide dose (1000 μg RHAMM-R3 peptide). The vaccine was given four times at a biweekly interval and GM-CSF was added for five days each vaccination. Similar to the patients vaccinated with 300 μg peptide only mild drug-related adverse events were observed such as erythema and induration of the skin. Immunomonitoring was performed using ELISpot assays for Interferon gamma and Granzyme B, tetramer-based flow cytometry and chromium release assays. Moreover, the frequency of regulatory T cells was quantified at different time points of vaccination. In this second cohort of patients treated with 1,000 μg peptide we detected specific immune responses in a lower frequency (4/9 patients) in contrast to patients in the 300 μg cohort (7/10 patients). In these patients with immune responses we found an increase of CD8+/HLA-A2/RHAMM-R3 tetramer+/CD45RA+/CCR7−/CD27−/CD28− effector T cells in flow cytometry in accordance with an increase of R3-specific CD8+ T cells in ELISpot assays. Two patients with positive immune responses showed a significant decrease of regulatory T cells. One patient without positive immune and clinical effects showed an increase of the frequency of regulatory T cells (5.03% to 15.9%). Three out of nine patients treated with 1,000 μg showed positive clinical effects: One patient with MDS RAEB-2 showed a reduction of leukemic blasts in the bone morrow to lower than 5%, one MDS patient achieved a normalization of the peripheral blood counts and one patient with multiple myeloma experienced a reduction of light chain in serum. The patients in the 300 μg cohort showed also a higher frequency of positive clinical effects (5 out of 10 patients). Taken together, RHAMM-R3 peptide vaccination induced both immunological and clinical responses. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematological malignancies. However, higher doses of peptide do not improve the frequency and intensity of immune responses in this clinical trial.

Cellular Immune Responses To Vector In a Gene Therapy Trial For Hemophilia B Using An AAV8 Self-Complementary Factor IX Vector

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 717-717
Author(s):  
Etiena Basner-Tschakarjan ◽  
Federico Mingozzi ◽  
Yifeng Chen ◽  
Amit Nathwani ◽  
Edward Tuddenham ◽  
...  
Keyword(s):  
Immune Response ◽  
T Cells ◽  
T Cell ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Factor Ix ◽  
High Dose ◽  
Advisory Committees ◽  
Ifn Γ ◽  
Cellular Immune

Abstract In a clinical study of gene transfer for hemophilia B an adeno-associated virus vector serotype 8 (AAV8) expressing a self-complementary liver-specific expression cassette for the factor IX (FIX) transgene was administered intravenously in ten affected subjects. The results of the first part of the study have been published (NEJM 365:2357-65, 2011). In this abstract we present the immunomonitoring data, using Interferon-gamma (IFN-γ) ELISpot and polyfunctional T cell analysis of peripheral blood mononuclear cells (PBMCs) to monitor cellular immune responses to vector capsid and to Factor IX. We have previously shown that the cellular immune response was directed solely towards AAV capsid epitopes, not FIX, and that the response was dose-dependent. Out of six subjects infused in the high dose cohort (2x1012vg/kg), 4/6 manifested a minor rise in liver enzyme levels and detection of capsid-specific T cell reactivitiy in the ELISpot assay at ∼7-10 weeks post vector infusion. Maximum results on IFN- γ ELISpots ranged from 200-500 sfu/million cells. In two of these cases a modest decline in FIX level also occurred. Prompt initiation of prednisolone reversed these effects and rescued FIX levels. The remaining two subjects infused at the high dose, showed no rise in liver enzyme levels at any time point. However capsid reactive T cells were detectable in one subject as early as one to two weeks after vector infusion in peripheral blood by IFN-γ ELISpot assay, while no activation at all was detected in the other subject, possibly due to low cell recovery and viability of the cells. A similar immune response profile, with early detection of activated T cells but no rise in liver enzymes, was also observed in both subjects in the intermediate dose cohort in the first part of this study. Polyfunctional T cell analysis revealed concurrent Interleukin-2, Tumor necrosis factor-alpha and CD107a positivity in activated T cells at the peak of activation. Furthermore it showed that capsid-specific early T cell responses were detectable in the CD4+ T cell and later in the CD8+T cell compartment. Long-term immune monitoring of all subjects is ongoing. Importantly in one of the first two subjects treated at the high dose, capsid reactive T cells were detected by ELISpot 1.5 years after gene transfer; these cells were not detected in the other subject in whom long-term follow-up samples are available. Of note, capsid-reactive T cells were also seen at late time points (>1 year after infusion) in a middle dose subject and a low dose subject. Despite detectable T cell reactivity towards the AAV capsid in the peripheral blood FIX expression remained stable, suggesting that there is a short window of time during which transduced hepatocytes present a target for cytotoxic T cells, and that T cell positivity after this window is without any clinical consequences. In conclusion, for this scAAV8 vector there appears to be a critical threshold vector dose for a clinically detectable immune response, starting at 2x1012 vg/kg. The clinically detectable response occurred in four out of six subjects so far, and was manifest within a critical time interval of 7-10 weeks post infusion. The capsid-specific response was polyfunctional and detected in CD4+ and CD8+T cells in peripheral blood. It is important to note that not all subjects treated at the high dose developed an immune response. However, given the limited dataset, it is not yet possible to define predictive parameters, e.g. HLA type of a subject, for an immune response. Continued monitoring and future studies with more subjects will be necessary to confirm the presented findings, in particular time and rate of occurrence of a cellular response as well as successful treatment with a short course of Prednisolon. Disclosures: Tuddenham: Pfizer: Consultancy. Reiss:Hemophilia of Georgia: Honoraria. High:BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; Genzyme, Inc.: Membership on an entity’s Board of Directors or advisory committees; Intrexon: Consultancy; Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Shire : Consultancy; Benitec: Consultancy; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; BioMarin: Consultancy; Alnylam Pharmaceuticals: Consultancy, Membership on an entity’s Board of Directors or advisory committees.

Regulatory T cells mediate resistance to radiotherapy in head and neck squamous cell carcinoma.

2019 ◽  
Vol 37 (8_suppl) ◽  
pp. 70-70 ◽  
Author(s):  
Ayman Oweida ◽  
Laurel Darragh ◽  
Shilpa Bhatia ◽  
David Raben ◽  
Lynn Heasley ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Tumor Growth ◽  
Head And Neck ◽  
Critical Role ◽  
Growth Delay ◽  
Functional Changes ◽  
Treg Depletion

70 Background: Head and neck tumors are highly enriched in regulatory T cells which dampen the response to radiotherapy by creating an immune-inhibitory microenvironment. We explored mechanisms of Treg infiltration and assessed their modulation by RT in murine models of HNSCC. Methods: Mechanisms of Treg infiltration were investigated in murine HNSCC tumors using whole genome sequencing and flow cytometry. Mice were treated with anti-CTLA-4, anti-CD-25 and/or anti-PD-L1 alone and in combination with RT. Tumor growth and survival were assessed. Flow cytometry was used to assess phenotypic and functional changes in intratumoral T cell populations. Multiplex ELISA was performed for assessment of cytokines. RNA Sequencing was performed to interrogate mechanisms of response and resistance to treatment. Results: Treatment with anti-CD-25 concurrently with RT led to significant tumor growth delay, enhanced T cell cytotoxicity, decreased Tregs and improved survival. In contrast CTLA-4 blockade did not affect tumor growth or survival. Treg depletion induced an influx of CD8 and CD4 T cells when combined with RT. In addition, Treg depletion in combination with RT transformed myeloid populations decreasing M2 macrophages and MDSCs and increasing M1 macrophages. Mechanistically, tumors secrete CCL20, a potent Treg chemoattractant responsible for creating a highly immunuosuppressive tumor microenvironment and potentially responsible for treatment resistance. Conclusions: These data reveal a critical role for regulatory T cells in mediating resistance to RT. Targeted depletion of Tregs represents an important mechanism of sensitizing tumors to RT. Our data support the design of clinical trials integrating targeted Treg inhibitors in the standard of care for cancer patients receiving RT.

scholarly journals Peripheral blood T cells in acute myeloid leukemia (AML) patients at diagnosis have abnormal phenotype and genotype and form defective immune synapses with AML blasts

Blood ◽  
2009 ◽  
Vol 114 (18) ◽  
pp. 3909-3916 ◽  
Author(s):  
Rifca Le Dieu ◽  
David C. Taussig ◽  
Alan G. Ramsay ◽  
Richard Mitter ◽  
Faridah Miraki-Moud ◽  
...  
Keyword(s):  
Gene Expression ◽  
Immune Response ◽  
T Cells ◽  
Acute Myeloid Leukemia ◽  
T Cell ◽  
Peripheral Blood ◽  
Myeloid Leukemia ◽  
Absolute Number ◽  
Immune Synapses ◽  
Acute Myeloid

Abstract Understanding how the immune system in patients with cancer interacts with malignant cells is critical for the development of successful immunotherapeutic strategies. We studied peripheral blood from newly diagnosed patients with acute myeloid leukemia (AML) to assess the impact of this disease on the patients' T cells. The absolute number of peripheral blood T cells is increased in AML compared with healthy controls. An increase in the absolute number of CD3+56+ cells was also noted. Gene expression profiling on T cells from AML patients compared with healthy donors demonstrated global differences in transcription suggesting aberrant T-cell activation patterns. These gene expression changes differ from those observed in chronic lymphocytic leukemia (CLL), indicating the heterogeneous means by which different tumors evade the host immune response. However, in common with CLL, differentially regulated genes involved in actin cytoskeletal formation were identified, and therefore the ability of T cells from AML patients to form immunologic synapses was assessed. Although AML T cells could form conjugates with autologous blasts, their ability to form immune synapses and recruit phosphotyrosine signaling molecules to the synapse was significantly impaired. These findings identify T-cell dysfunction in AML that may contribute to the failure of a host immune response against leukemic blasts.

scholarly journals Platelet Desialylation As a Predictive Marker in Childhood Immune Thrombocytopenia (ITP)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 221-221
Author(s):  
Leona Raskova Kafkova ◽  
Diana Brokesova ◽  
Zbynek Novak ◽  
Milan Raska ◽  
Dagmar Pospisilova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Immune Response ◽  
T Cells ◽  
Transcription Factors ◽  
T Cell ◽  
Immune Thrombocytopenia ◽  
Proper Function ◽  
Healthy Controls ◽  
Platelet Surface ◽  
Surface Bond

Background and aim: Immune thrombocytopenia (ITP) is the most common bleeding condition in children. Its prognosis is mostly superior, however, severe refractory disease remains diagnostic and therapeutic challenge. Low platelet counts (&lt;100× 109/L) are associated with increased platelet clearance by two parallel mechanisms: classical antibody-mediated pathway and a novel lectin-carbohydrate mediated pathway. The latter is based on platelet desialylation, where terminal sialic acids are cleaved from glycoconjugates, mainly glycoproteins (GPs), on the platelet surface. The loss of sialic acid enhances bond of the penultimate β-galactose to asialoglycoprotein receptors (ASGPRs, also called Ashwell-Morell receptors) on hepatocytes. Desialylated platelets are then captured and phagocytosed by ASGPR-expressing hepatocytes. Desialylation has been shown to be responsible for platelet destruction in many contexts, e.g., infection-related thrombocytopenia or clearance of senescent platelets. Loss of T-cell tolerance is another underlying mechanism in ITP; CD8+ regulatory T cells (Tregs) are able to inhibit overactive immune response and maintain immune homeostasis. Forkhead box P3 (FOXP3) and GATA3 are transcription factors crucial for development and proper function of Tregs limiting the Th2-type inflammatory response. Our aims were to distinguish contribution of the mentioned processes to ITP development and to characterize immune response in children with ITP during the course of disease (diagnosis, ongoing therapy, remission, refractory/persistent ITP). Patients and Methods: We examined 30 samples from 20 children with ITP (12 males, 8 females, age 3-17 years; 3 acute ITP, 17 chronic ITP) and 10 healthy controls (age 4-15). The degree of desialylation was determined by flow cytometry using FITC-labeled Ricinus communis agglutinin (RCA-I) specific for terminal galactose or N-acetylgalactosamine. Expression of platelet surface markers was given quantitatively as mean fluorescence intensity (MFI). Presence of platelet surface-bond antibodies (IgG, IgA and IgM) was examined by flow cytometry. Subpopulations of CD4+ and CD8+ T-cells were characterized based on intracellular expression of transcription factors T-bet (Th1 cells), GATA3 (Th2 cells), ROR gamma T (Th17 cells) and FOXP3 (for Tregs) using multicolor flow cytometry. Results: Patients with ITP showed significant increase in RCA-I reactivity in comparison with healthy controls (p&lt;0.001). Patients with newly diagnosed ITP showed the most aberrant sialylation (i.e., maximum desialylation) of platelet surface proteins. A decrease in desialylation intensity was noticeable as soon as at three days after therapy initiation. Sialylation levels returned to normal after one month of successful treatment and were similar to healthy controls in children with ITP remission. Platelet surface-bond immunoglobulins were increased in 10 (50%) patients independently on their sialylation level. We observed significant changes in T-cell subpopulations in ITP: T lymphocytes producing T-bet were decreased within both CD4+ and CD8+ populations. Percentage of CD4+ cells expressing ROR gamma T was also reduced. Proportions of cells expressing FOXP3 and GATA3 were decreased within the CD8+ but not within the CD4+ population. Conclusion: Our results highlight the importance of Fc-independent hepatic platelet clearance in ITP. Interindividual differences in ITP pathophysiology are reflected by treatment response and may improve therapeutic management and prognostication. E.g., intravenous immunoglobulins or splenectomy will be ineffective in patients with prevalent Fc-independent mechanisms, and contrarily, possibilities for novel targeted treatment (neuraminidase inhibitors) arise. Better understanding of immune-mediated processes involved in ITP pathogenesis may reduce adverse effects of immunosuppressive therapy and considerably improve quality of life in patients with ITP. Supported by: MH CZ - DRO (FNOl, 00098892), Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868) and Ministry of Education, Youth and Sports OPVVV CEREBIT CZ.02.1.01/0.0/0.0/16_025/0007397. Disclosures No relevant conflicts of interest to declare.

Cellular side of acquired immunity (T cells)

2013 ◽  
Author(s):  
Assia Eljaafari ◽  
Pierre Miossec
Keyword(s):  
Immune Response ◽  
T Cells ◽  
Immune System ◽  
Disease Process ◽  
T Cell Response ◽  
Th2 Cells ◽  
Cell Mediated Immunity ◽  
Immune Effector ◽  
Effector Cells ◽  
Th22 Cells

The adaptive T-cell response represents the most sophisticated component of the immune response. Foreign invaders are recognized first by cells of the innate immune system. This leads to a rapid and non-specific inflammatory response, followed by induction of the adaptive and specific immune response. Different adaptive responses can be promoted, depending on the predominant effector cells that are involved, which themselves depend on the microbial/antigen stimuli. As examples, Th1 cells contribute to cell-mediated immunity against intracellular pathogens, Th2 cells protect against parasites, and Th17 cells act against extracellular bacteria and fungi that are not cleared by Th1 and Th2 cells. Among the new subsets, Th22 cells protect against disruption of epithelial layers secondary to invading pathogens. Finally these effector subsets are regulated by regulatory T cells. These T helper subsets counteract each other to maintain the homeostasis of the immune system, but this balance can be easily disrupted, leading to chronic inflammation or autoimmune diseases. The challenge is to detect early changes in this balance, prior to its clinical expression. New molecular tools such as microarrays could be used to determine the predominant profile of the immune effector cells involved in a disease process. Such understanding should provide better therapeutic tools to counteract deregulated effector cells.

Rapamycin Generated Murine Th2/Tc2 Cells Potently Abrogate Fully MHC-Mismatched Hematopoietic Stem Cell (HSC) Graft Rejection.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 192-192
Author(s):  
Jacopo Mariotti ◽  
Jason Foley ◽  
Todd Borenstein ◽  
Soo Han ◽  
Daniel H. Fowler
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Graft Rejection ◽  
Fas Ligand ◽  
Absolute Number ◽  
Th2 Cells ◽  
Ifn Gamma ◽  
Donor T Cells

Abstract We previously showed that costimulated Th2/Tc2 cells abrogate graft rejection. In recent experiments, we found that ex vivo rapamycin generates Th2 cells (Th2R cells) that more potently prevent GVHD relative to control Th2 cells. We thus hypothesized that rapamycin may improve the ability of Th2/Tc2 cells to abrogate graft rejection. To test this hypothesis, we utilized a recently defined model of rejection involving lethal host irradiation and subsequent quantitative host T cell addback [B6(H-2b) into BALB/c(H-2d), TBI:10.5 Gy; 0.1x10^6 host T cell addback]. To generate stem-cell enriched allografts, donor mice were treated with G-CSF (5 microgram/d for 5 d) and resultant spleen cells were expanded for 6 d in rhTPO, rmSCF and rhFLT3L: relative to input cells, the final product was T cell depleted (%CD3 reduced from 9.7±0.6% to 0.4±0.03%) and stem-cell enriched by KLS analysis (%c-kit+Lin-Sca-1+ increased from 5.7±1.8% to 49.08±2.8%) and by functional analysis (%side population increased from 0.24±0.04% to 1.7±0.2). In an initial experiment that did not involve host T cell addback, we determined that 10x10^6 of the HSC product was radioprotective in 100% of recipients (10/10 subjects, 90 d follow-up) and yielded 100% donor chimerism without clinical GVHD. To generate donor Th2/Tc2 and Th2/Tc2R cells, donor T cells were costimulated with anti-CD3/CD28 coated beads and expanded in media supplemented with rmIL-4, rhIL-2, rhIL-7 either without or with high dose rapamycin (10 micromolar). Host-vs-graft (HVG) responses were quantified at d 5 post transplant by the following method: (a) spleen cell harvest and enumeration; (b) 24 h host (syngeneic) or donor (allogeneic) dendritic cell stimulation; (c) cell-surface flow cytometry with anti-CD4, anti-CD8 and anti-host (H-2d) antibodies; (d) Miltenyi IFN-gamma cytokine capture flow cytometry; and (e) calculation of absolute number of host anti-donor alloreactive CD4+ and CD8+ T cells per spleen. HSC transfer without Th2/Tc2 cells induced robust CD4- and CD8-mediated HVG responses (Fig.1, cohort 2>cohort 1; p<0.001). HSC transfer augmented with donor Th2/Tc2R cells fully abrogated HVG responses; in marked contrast, HSC transfer augmented with control Th2/Tc2 cells only partially reduced HVG responses. Rapamycin generated Th2/Tc2 cells were more potent than control Th2/Tc2 cells with respect to abrogation of both CD4- and CD8-mediated HVG responses (p<0.05). The mechanism of Th2/Tc2R cells abrogation of HVG responses involved both CD4+Th2R and CD8+Tc2R cell components, and did not require IL-4, perforin, or fas ligand. At day 90 post transplant, Th2/Tc2R cell recipients had nominal GVHD (<5% weight loss) and consistent alloengraftment (>99% chimerism, 9 of 10 recipients; rejection, 1 of 10 recipients); in marked contrast, control Th2/Tc2 cell recipients had either graft rejection (9 of 10 recipients) or mixed chimerism (1 of 10 recipients). In conclusion, ex vivo rapamycin generates donor Th2/Tc2 cells that potently abrogate HVG responses and HSC graft rejection through a mechanism that involves CD4+Th2 and CD8+Tc2 cells and non-classical molecular effectors. Figure 1: Donor Th2/Tc2R Cell Modulation of HVG Responses. B6-into-BALB/c transplantation was performed (10 x 106 donor HSC; 10 x 106 donor T cells; 1 x 106 host T cells; 1050 cGy XRT; n=10 mice per cohort). Mice were killed at d 5 post-SCT, and absolute number of host anti-donor alloreactive CD4 and CD8 Cells was determined by IFN-gamma cytokine capture flow cytometry. * indicates p<0.05 and ** indicates p<0.001 (student’s t-test); each cohort compared with cohort #2. Abbreviations: KO, Knockout, PFN, perfocin, FASL, fas ligand. Figure 1:. Donor Th2/Tc2R Cell Modulation of HVG Responses. B6-into-BALB/c transplantation was performed (10 x 106 donor HSC; 10 x 106 donor T cells; 1 x 106 host T cells; 1050 cGy XRT; n=10 mice per cohort). Mice were killed at d 5 post-SCT, and absolute number of host anti-donor alloreactive CD4 and CD8 Cells was determined by IFN-gamma cytokine capture flow cytometry. * indicates p<0.05 and ** indicates p<0.001 (student’s t-test); each cohort compared with cohort #2. Abbreviations: KO, Knockout, PFN, perfocin, FASL, fas ligand.

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