Antiviral Activity of Shiga Toxin 1: Suppression of Bovine Leukemia Virus-Related Spontaneous Lymphocyte Proliferation

ABSTRACT Human infections with Shiga toxin (Stx)-producing Escherichia coli (STEC) cause hemorrhagic colitis. The Stxs belong to a large family of ribosome-inactivating proteins (RIPs) that are found in a variety of higher plants and some bacteria. Many RIPs have potent antiviral activity for the plants that synthesize them. STEC strains, both virulent and nonvirulent to humans, are frequently isolated from healthy cattle. Interestingly, despite intensive investigations, it is not known why cattle carry STEC. We tested the hypothesis that Stx has antiviral properties for bovine viruses by assessing the impact of Stx type 1 (Stx1) on bovine peripheral blood mononuclear cells (PBMC) from cows infected with bovine leukemia virus (BLV). PBMC from BLV-positive animals invariably displayed spontaneous lymphocyte proliferation (SLP) in vitro. Stx1 or the toxin A subunit (Stx1A) strongly inhibited SLP. Toxin only weakly reduced the pokeweed mitogen- or interleukin-2-induced proliferation of PBMC from normal (BLV-negative) cows and had no effect on concanavalin A-induced proliferation. The toxin activity in PBMC from BLV-positive cattle was selective for viral SLP and did not abrogate cell response to pokeweed mitogen- or interleukin-2-induced proliferation. Antibody to virus or Stx1A was most effective at inhibiting SLP if administered at the start of cell culture, indicating that both reagents likely interfere with BLV-dependent initiation of SLP. Stx1A inhibited expression of BLV p24 protein by PBMC. A well-defined mutant Stx1A (E167D) that has decreased catalytic activity was not effective at inhibiting SLP, suggesting the inhibition of protein synthesis is likely the mechanism of toxin antiviral activity. Our data suggest that Stx has potent antiviral activity and may serve an important role in BLV-infected cattle by inhibiting BLV replication and thus slowing the progression of infection to its malignant end stage.

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Intestinal Shiga Toxin-Producing Escherichia coli Bacteria Mitigate Bovine Leukemia Virus Infection in Experimentally Infected Sheep

Infection and Immunity ◽

10.1128/iai.74.5.2906-2916.2006 ◽

2006 ◽

Vol 74 (5) ◽

pp. 2906-2916 ◽

Cited By ~ 13

Author(s):

Witold A. Ferens ◽

Rowland Cobbold ◽

Carolyn J. Hovde

Keyword(s):

Escherichia Coli ◽

Antiviral Activity ◽

Shiga Toxin ◽

Bovine Leukemia Virus ◽

Mononuclear Cells ◽

Leukemia Virus ◽

Average Weight ◽

E Coli ◽

Infected Cells ◽

The Impact

ABSTRACTRuminants often carry gastrointestinal Shiga toxin (Stx)-producingEscherichia coli(STEC). Stxs belong to a large family of ribosome-inactivating proteins (RIPs), found in many plants and some bacteria. Plant RIPs, secreted into extracellular spaces, limit the spread of viruses through plant tissues by penetrating and killing virally infected cells. Previously, we showed Stx activity against bovine leukemia virus (BLV)-infected cells in vitro and hypothesized that STEC bacteria have antiviral activity in ruminant hosts. Here, we investigated the impact of STEC on the initial phases of BLV infection in sheep. Sheep were treated with biweekly oral doses ofE. coliO157:H7 (an STEC) or an isogenicstxmutant strain. A different group of sheep were similarly treated with five naturally occurring ovine STEC isolates orstx-negativeE. coli. Intestinal STEC bacteria were enumerated and identified by standard fecal culture and DNA hybridization. Oral STEC treatment did not always result in carriage of STEC, although many animals consistently presented with >104CFU/g feces. BLV viremia was assessed by spontaneous lymphocyte proliferation (SLP) in cultures of blood mononuclear cells and by syncytium formation in cocultures of the same with F-81 indicator cells. SLP was lower (P< 0.05) and syncytia were fewer (P < 0.05) in STEC-treated sheep than in untreated sheep. Both lower SLP and fewer syncytia positively correlated with fecal STEC numbers. Average weight gain post-BLV challenge was higher in STEC-treated sheep than in untreated sheep (P< 0.05). These results support the hypothesis that in ruminants, intestinal STEC bacteria have antiviral activity and mitigate BLV-induced disease.

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Regulation of Bovine Leukemia Virus taxand pol mRNA Levels by Interleukin-2 and -10

Journal of Virology ◽

10.1128/jvi.73.10.8427-8434.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8427-8434 ◽

Cited By ~ 13

Author(s):

Dohun Pyeon ◽

Gary A. Splitter

Keyword(s):

Bovine Leukemia Virus ◽

Mononuclear Cells ◽

Leukemia Virus ◽

Interleukin 2 ◽

Disease Stage ◽

Mrna Levels ◽

Soluble Factor ◽

Early Disease ◽

P24 Protein ◽

Disease Stages

ABSTRACT Recently, particular cytokines have been identified to affect progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma interferon increased in peripheral blood mononuclear cells (PBMCs) from animals with early disease and decreased in PBMCs from animals with late disease stages of bovine leukemia virus (BLV) infection. In contrast, IL-10 increased with disease progression. To examine the effects of these cytokines on BLV expression, BLV tax andpol mRNA and p24 protein were quantified by competitive PCR and immunoblotting, respectively. IL-10 inhibited BLV taxand pol mRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes and/or macrophages (monocyte/macrophages). To determine whether these factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between monocyte/macrophages and nonadherent PBMCs was blocked. BLVtax and pol mRNA levels increased in transwell cultures similar to cultures containing nonseparated cells, and IL-10 addition inhibited the increase of BLV tax andpol mRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLV tax and pol mRNA and p24 protein production. Thus, IL-10 production by BLV-infected animals with late stage disease may serve to control BLV mRNA levels, while IL-2 may increase BLV mRNA in the early disease stage. To determine a correlation between cell proliferation and BLV expression, the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10 production in late disease stages suppresses BLV mRNA levels, while IL-2-activated immune responses stimulate BLV expression by BLV-infected B cells.

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Shiga Toxin 1 Targets Bovine Leukemia Virus-Expressing Cells

Infection and Immunity ◽

10.1128/iai.72.3.1837-1840.2004 ◽

2004 ◽

Vol 72 (3) ◽

pp. 1837-1840 ◽

Cited By ~ 12

Author(s):

Witold A. Ferens ◽

Luke J. Grauke ◽

Carolyn J. Hovde

Keyword(s):

Shiga Toxin ◽

Bovine Leukemia Virus ◽

Direct Evidence ◽

Mononuclear Cells ◽

Leukemia Virus ◽

Peripheral Blood Mononuclear ◽

Electron Microscopy Analysis ◽

A Subunit ◽

Microscopy Analysis

ABSTRACT Direct evidence that Escherichia coli Shiga toxin (Stx) acts against bovine leukemia virus (BLV)-expressing cells was obtained. The active A subunit of Stx type 1 (StxA1) targeted a selected population of permeable cells expressing BLV and inhibited BLV replication in a culture of bovine peripheral blood mononuclear cells. Cells were cultured with and without StxA1, and at various times cells expressing BLV were identified by being stained with MW1 monoclonal antibody specific for the BLV protein gp51. Before culture, permeable cells were tagged by uptake of one of the following: acetoxymethyl of 2′,7′-bis-(2-carboxyethyl)-5-(and 6)-carboxyfluorescein (BCECF), BCECF conjugated to 70-kDa dextran, or 70-kDa dextran conjugated to fluorescein. The tagged cells costaining with anti-gp51 were selectively eliminated in StxA1-treated cultures. Electron microscopy analysis of purified B lymphocytes showed sharply reduced numbers of BLV particles in StxA1-treated cultures.

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Natural Infection of Dairy Cows with Bovine Leukemia Virus Affects Immunoglobulin Levels in Saliva and Serum but Not Milk

Pathogens ◽

10.3390/pathogens10070907 ◽

2021 ◽

Vol 10 (7) ◽

pp. 907

Author(s):

Monika Dziuba ◽

Vickie J. Ruggiero ◽

Catherine Wilson ◽

Paul C. Bartlett ◽

Paul M. Coussens

Keyword(s):

Pilot Study ◽

Dairy Cows ◽

Bovine Leukemia Virus ◽

Leukemia Virus ◽

Natural Infection ◽

Total Serum ◽

Serum Samples ◽

Retroviral Infection ◽

Serum Iga ◽

The Impact

Bovine leukemia virus (BLV) is a retroviral infection that disrupts the immune function of infected animals. It is widespread among U.S. dairy cattle. In this pilot study, the average total IgA and IgM concentrations in milk, saliva, and serum samples from BLV ELISA-positive (ELISA+) dairy cows were compared against samples from BLV ELISA-negative (ELISA−) cows using the Kruskal–Wallis test (with ties). The results from ELISA+ cows were also stratified by lymphocyte count (LC) and proviral load (PVL). In milk and saliva from ELISA+ cows, the average total IgA and IgM concentrations were decreased compared to ELISA− cows, although this was only statistically significant for saliva IgM in cows with low PVL (p = 0.0424). Numerically, the average total IgA concentrations were 33.6% lower in milk and 23.7% lower in saliva, and the average total IgM concentrations were 42.4% lower in milk and 15.5% lower in saliva. No significant differences were observed in the total serum IgA concentrations, regardless of PVL and LC. The total serum IgM from ELISA+ cows was significantly decreased (p = 0.0223), with the largest decreases occurring in the highest PVL and LC subgroups. This pilot study is a first step in investigating the impact of BLV on mucosal immunity and will require further exploration in each of the various stages of disease progression.

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Influence of the Bovine Leukemia Virus on the Immunological Activity by the Neutrophilic Function

Acta Scientiae Veterinariae ◽

10.22456/1679-9216.102520 ◽

2020 ◽

Vol 48 ◽

Author(s):

Vinícius Bigolin Narciso ◽

Silvana Giacomini Collet ◽

Lilian Kolling Girardini ◽

Fernando Nogueira Souza ◽

Alcione Santa Catarina ◽

...

Keyword(s):

Oxidative Metabolism ◽

Bovine Leukemia Virus ◽

Leukemia Virus ◽

Latency Period ◽

Persistent Lymphocytosis ◽

Phagocytic Capacity ◽

Significant Difference ◽

Zymosan Particle ◽

The Impact ◽

Hematological Alterations

Background: Bovine leukemia virus (VLB) is an oncogenic deltaretrovirus associated with the development of persistent lymphocytosis (LP) and lymphosarcomas in cattle. LP is characterized by chronic elevation of the number of circulating lymphocytes, in the case of B lymphocytes. Several studies have described functional changes in various leukocyte populations in both blood and milk in VLB-infected animals. The impact of some chronic diseases of low lethality is aggravated by the emergence of comorbidities.The objective of the present study was to evaluate the oxidative metabolism and neutrophil phagocytosis of bovines of the Holtein breed naturally infected with the bovine leukemia virus (VLB).Materials, Methods & Results: In this study, 20 cows were divided into three groups: (NG) seven non-seroreagent animals for VLB and without hematological alterations; (GAL) eight seroreagent animals for VLB and without hematological alterations; and (GLP) five seroreagent animals for VLB with persistent lymphocytosis (LP). The oxidative metabolism of neutrophils was determined by the tetrazolium nitroblast reduction test stimulated or not with Zymosan particles. The percentage of neutrophils that phagocytosed Zymosan particle (s) was also evaluated. The data were initially evaluated for normality and homoscedasticity by the Shapiro-Wilk test. Then the ANOVA test followed by the Student-Newman-Keuls test was applied for the comparison between the NG, GAL and GLP animals. Comparison between the NG animals and the seroreagent animals for the VLB (GVLB) was also performed through the unpaired Student's t-test. The value of P < 0.05 was considered significant. No significant differences were observed in oxidative neutrophil metabolism in stimulated and non-stimulated samples with Zymosan particles nor in the percentage of neutrophils that phagocytosed Zymosan particle (s) among the three experimental groups. However, as no differences were observed between the seroreagent animals for VLB with and without LP, we chose to divide the animals into only two experimental, non-seroreagent and seroreagent groups for VLB. Thus, when non-seroreagent animals for the VLB were compared with the seroreagent animals for the VLB, which corresponds to the GAL and GLP animals, a significant difference was observed in relation to the oxidative metabolism by neutrophils stimulated with Zymosan particles.Discussion: Some viral diseases are often associated with increased susceptibility to new infections and several studies have evaluated the role of peripheral blood mononuclear cells in VLB infection, but few studies have investigated neutrophil function. Some authors, when evaluating phagocytic capacity and oxidative metabolism, respectively, of blood leukocytes from VLB-infected animals, observed that VLB-infected animals displaying LP had lower phagocytic capacity and lower production of Reactive Oxygen Species (ROS). Some studies have shown that oxygen consumption by neutrophils was higher in experimentally infected sheep by VLB after 15 weeks of challenge, but this species is not a natural host of the virus, since transmission does not occur between sheep and cattle and the pathogenesis of infection by VLB is more acute in sheep, a result of the lower latency period for LP development. Other authors, when evaluating the interference of VLB in milk leukocytes, concluded that VLB-infected animals show lower intensity of intracellular ROS production by flow cytometry in VLB-infected animals, especially animals expressing LP, despite the fact that percentage of milk neutrophils that produced ROS did not differ between groups. It can be concluded that VLB interferes in neutrophilic function with possible implications for the health of VLB-infected animals and may favor secondary infections.

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Immunologic correlates of spontaneous lymphocyte proliferation in human T-lymphotropic virus infection

Blood ◽

10.1182/blood.v78.1.169.169 ◽

1991 ◽

Vol 78 (1) ◽

pp. 169-174 ◽

Cited By ~ 14

Author(s):

HE Prince ◽

H Lee ◽

ER Jensen ◽

P Swanson ◽

D Weber ◽

...

Keyword(s):

Lymphocyte Proliferation ◽

Mononuclear Cells ◽

Interleukin 2 ◽

Serum Levels ◽

Control Group ◽

Relative Distribution ◽

T Lymphotropic Virus ◽

Soluble Cd25 ◽

Spontaneous Proliferation ◽

Antibody Levels

Abstract Previously we showed that mononuclear cells from about half of human T- lymphotropic virus (HTLV)-seropositive persons exhibit spontaneous proliferation in vitro. We sought to determine if proliferation was associated with other immunologic changes characteristic of HTLV infection. The parameters assessed were (1) percentages of lymphocytes expressing CD4 and/or CD25 (interleukin-2 receptor), (2) serum levels of soluble CD25, (3) serostatus for other viruses, (4) anti-HTLV antibody levels, and (5) HTLV type determined by polymerase chain reaction or serologic reactivity with type-specific peptides. The proliferation+ HTLV (PROL+) group, proliferation HTLV (PROL-) group, and control group showed similar percentages of CD4+, CD25+, and CD4+CD25+ lymphocytes; serum levels of soluble CD25 were also similar. Antibodies to cytomegalovirus, hepatitis B core, and hepatitis C were present in similar proportions of PROL+ and PROL+ groups. However, a significant association was found between spontaneous proliferation and anti-HTLV antibody levels; sera from 67% of PROL+ persons, but only 18% of PROL- persons, required dilution to yield absorbance values within the linear range of the anti-HTLV antibody assay. In the PROL+ group, persons whose sera required the most dilution had proliferative responses significantly higher than those whose sera required no dilution. The PROL+ and PROL groups were similar with regard to the relative distribution of HTLV-I and HTLV-II infection. These findings indicate that HTLV-related spontaneous lymphocyte proliferation is related to levels of circulating anti-HTLV antibodies, and characterizes both HTLV-I and HTLV-II infection.

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Bovine Leukemia Virus and Mycobacterium avium subsp. paratuberculosis Are Not Associated with Shiga Toxin–Producing Escherichia coli Shedding in Cattle

Journal of Food Protection ◽

10.4315/0362-028x.jfp-16-090 ◽

2016 ◽

Vol 80 (1) ◽

pp. 86-89

Author(s):

CRISTINA VENEGAS-VARGAS ◽

SHANNON D. MANNING ◽

PAUL M. COUSSENS ◽

JONATHAN A. ROUSSEY ◽

PAUL BARTLETT ◽

...

Keyword(s):

Escherichia Coli ◽

Mycobacterium Avium ◽

Shiga Toxin ◽

Bovine Leukemia Virus ◽

Leukemia Virus ◽

Mycobacterium Avium Subsp ◽

Antibody Detection ◽

P Value ◽

Beef Herds ◽

Mycobacterium Avium Subsp Paratuberculosis

ABSTRACT Bovine leukemia virus (BLV) is a retrovirus that causes enzootic bovine leukosis in cattle, and Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of Johne's disease in cattle. Both diseases are chronic in nature and can lead to the disruption of normal immunological or physiological processes. Cattle are the major reservoir of Shiga toxin–producing Escherichia coli (STEC), a cause of foodborne illness in humans. We tested the hypothesis that cattle infected with BLV or MAP are more likely to shed STEC. We conducted a cross-sectional study during the summers of 2011 and 2012 in 11 Michigan cattle herds. A fecal sample from each animal was collected for STEC culture, and multiplex PCR for stx1, stx2, and eaeA was used to screen suspect colonies for STEC confirmation. Antibody detection enzyme-linked immunosorbent assays for BLV and MAP were used to screen serum from each animal. Flow cytometry was used to quantify the percentage of lymphocytes, monocytes, and neutrophils in a subsample (n =497) of blood samples. Of the animals sampled, 34.9% were BLV positive, 2.7% were MAP positive, and 16% were shedding STEC. Cattle in the dairy herds had a higher frequency of BLV and MAP than did those in beef herds, but more cattle in beef herds were shedding STEC. Neither BLV nor MAP was associated with STEC shedding (P values of 0.6838 and 0.3341, respectively). We also observed no association between STEC status and the percentage of neutrophils (P value of 0.3565), lymphocytes (P value of 0.8422), or the lymphocyte-to-monocyte ratio (P value of 0.1800). Although controlling both BLV and MAP is important for overall herd health and productivity, we found no evidence that controlling BLV and MAP has an impact on STEC shedding in cattle.

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Involvement of Glutathione as a Mechanism of Indirect Protection against Spontaneous Ex Vivo Apoptosis Associated with Bovine Leukemia Virus

Journal of Virology ◽

10.1128/jvi.78.12.6180-6189.2004 ◽

2004 ◽

Vol 78 (12) ◽

pp. 6180-6189 ◽

Cited By ~ 3

Author(s):

Teresa Sanchez Alcaraz ◽

Pierre Kerkhofs ◽

Michal Reichert ◽

Richard Kettmann ◽

Luc Willems

Keyword(s):

Cell Death ◽

B Lymphocytes ◽

Bovine Leukemia Virus ◽

Mononuclear Cells ◽

Phorbol Esters ◽

Ex Vivo ◽

Leukemia Virus ◽

Infected Sheep ◽

Host Cells ◽

Spontaneous Apoptosis

ABSTRACT Viruses have developed strategies to counteract the apoptotic response of the infected host cells. Modulation of apoptosis is also thought to be a major component of viral persistence and progression to leukemia induced by retroviruses like human T-lymphotropic virus type 1 (HTLV-1) and bovine leukemia virus (BLV). Here, we analyzed the mechanism of ex vivo apoptosis occurring after isolation of peripheral blood mononuclear cells from BLV-infected sheep. We show that spontaneous apoptosis of ovine B lymphocytes requires at least in part a caspase 8-dependent pathway regardless of viral infection. Cell death is independent of cytotoxic response and does not involve the tumor necrosis factor alpha/NF-κB/nitric oxide synthase/cyclooxygenase pathway. In contrast, pharmaceutical depletion of reduced glutathione (namely, γ-glutamyl-l-cysteinyl-glycine [GSH]) by using ethacrynic acid or 1-pyrrolidinecarbodithioic acid specifically reverts inhibition of spontaneous apoptosis conferred indirectly by protective BLV-conditioned media; inversely, exogenously provided membrane-permeable GSH-monoethyl ester restores cell viability in B lymphocytes of BLV-infected sheep. Most importantly, intracellular GSH levels correlate with virus-associated protection against apoptosis but not with general inhibition of cell death induced by polyclonal activators, such as phorbol esters and ionomycin. Finally, inhibition of apoptosis does not correlate with the activities of GSH peroxidase and GSH reductase. In summary, our data fit into a model in which modulation of the glutathione system is a key event involved in indirect inhibition of apoptosis associated with BLV. These observations could have decisive effects during therapeutic treatment of δ-retroviral pathogenesis.

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Cytokine Production in Cell Culture by Peripheral Blood Mononuclear Cells from Immunocompetent Hosts

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.5.1.78-81.1998 ◽

1998 ◽

Vol 5 (1) ◽

pp. 78-81 ◽

Cited By ~ 50

Author(s):

Rohit K. Katial ◽

Doris Sachanandani ◽

Carolyn Pinney ◽

Michael M. Lieberman

Keyword(s):

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Interleukin 2 ◽

Pokeweed Mitogen ◽

Peripheral Blood Mononuclear ◽

Necrosis Factor Alpha ◽

Blood Mononuclear Cells ◽

Factor Alpha

ABSTRACT The production of interleukin 2 (IL-2) gamma interferon, IL-4, tumor necrosis factor alpha (TNF-α), TNF-β, IL-5, and IL-10 in vitro by peripheral blood mononuclear cells cultured from healthy immunocompetent subjects after mitogen stimulation was determined. The mitogens used were concanavalin A, phytohemagglutinin, pokeweed mitogen, and Staphylococcus aureus Cowen. The results obtained provide a normal range for the production of these cytokines under specified conditions in vitro.

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Interleukin-2 (hrIL2) Primed T cells display Reduced Alloproliferative Capacity In Vitro Due To Induction Of FOXP3+ Regulatory Cells

Blood ◽

10.1182/blood.v122.21.5433.5433 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5433-5433

Author(s):

Dimitra Kokkinou ◽

Panagiota Stamou ◽

Angeliki Vittoraki ◽

Anne-Lise De Lastic ◽

Spyros Chondropoulos ◽

...

Keyword(s):

T Cells ◽

Low Dose ◽

Mononuclear Cells ◽

Conflicts Of Interest ◽

Interleukin 2 ◽

Regulatory Cells ◽

Transplant Tolerance ◽

Immune Exhaustion ◽

The Impact

Abstract Introduction Prophylactic donor lymphocyte infusions (pDLI) after allogeneic transplantation contribute to immune restoration and reduce viral infections. Furthermore, we have recently shown that pDLI in patients with high risk leukemia significantly reduces the relapse rate, however, they were associated with a relatively high incidence of Graft versus Host Disease (GvHD)(BBMT 2013;19:75-81). Strategies to minimize GvHD without compromising the effect of pDLI against leukemia are needed. IL2 plays dual role in immune responses, contributing to both the generation of effector T cells and the maintenance of regulatory T cells (Tregs). Recently, low dose interleukin-2 (IL-2) therapy has been advanced as a potential immune modulator able to modulate the immune response to aid transplant tolerance and to suppress GvHD through expansion of Tregs (N Engl J Med. 2011; 2055-66). We investigated the impact of priming DLI with low dose IL2 on the proliferative responses to allo-stimulation in vitro. Methods CD3+ T cells purified from healthy individuals by MACS negative selection were primed (p-T cells) or unprimed (np-T cells), with or without (control) 100 U/ml hrIL2 (Proleukin, Novartis) for 7 days. Composition of T-cell cultures was analyzed by flow cytometry for: a) the percentage of T regulatory cells (CD4+/CD25high/Foxp3+/Helios+, b) their differentiation (CD28/CD27), c) their immune exhaustion (Programmed cell death 1, PD1). In vitro alloproliferative capacity of the p-T cells was analyzed with CFSE cell proliferation assay by using them as responder cells in mixed lymphocyte cultures (MLC), with irradiated allo-PB mononuclear cells as stimulators. Results In vitro priming of T-cells with IL-2 (p-T cells) in contrast to np-T or control cells: 1) increase the numbers of CD4+CD25highFoxp3+/Helios+ cells (n=8, 3.3%±0.7 mean±SEM vs 1.01%±0.22, p=0.004 και 1.4%±0.42, p=0.006). Increased levels of Foxp3 expression was also confirmed by Real Time PCR (n=2,1.25AU±0.15 vs 0.29AU±0.04, p=0.028 και 0.26AU±0.07, p=0.024). 2) did not affect the proportion of CD28+/CD27+ non late-differentiated cells (n=3, 60%±0.15 vs61%±0.04, p=0.91 και 59%±0.08, p=0.024). 3) did not cause immune exhaustion through PD1 expression (n=6, 13.3%±1.9 vs 8.1%±2.1, p=0.76, και 14%±2.2, p=0.68). 4) significantly decreases their response rate to allo-stimulus in MLC (n=8, 45%±0.5 vs65%±0.2, p=0.006 και 64%±0.2, p=0.008). The p-T cells regained their alloproliferative capacity after FACS-sorting removal of CD4+/CD25high Tregs. Conclusions Our results show that ex vivo priming of T cells with low dose of IL-2 reduces their in vitro alloproliferative capacity. This reduction is not due to late differentiation or immune – exhaustion of T cells but to selective induction of Foxp3+ cells with immunomodulatory properties in the culture. It remains to be seen whether IL2-primed DLI is safe and effective in transplant patients. Disclosures: No relevant conflicts of interest to declare.

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