scholarly journals Combined Deficiency of the Melanocortin 5 Receptor and Adenosine 2A Receptor Unexpectedly Provides Resistance to Autoimmune Disease in a CD8+ T Cell-Dependent Manner

2021 ◽  
Vol 12 ◽  
Author(s):  
Trisha McDonald ◽  
Fauziyya Muhammad ◽  
Kayleigh Peters ◽  
Darren J. Lee
Keyword(s):  
T Cells ◽  
T Cell ◽  
Autoimmune Disease ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Subsets ◽  
Dependent Manner ◽  
Knock Out ◽  
Adenosine 2A Receptor ◽  
Knock Out Mouse

Regulatory immunity that provides resistance to relapse emerges during resolution of experimental autoimmune uveitis (EAU). This post-EAU regulatory immunity requires a melanocortin 5 receptor (MC5r)-dependent suppressor antigen presenting cell (APC), as shown using a MC5r single knock-out mouse. The MC5r-dependent APC activates an adenosine 2A receptor (A2Ar)-dependent regulatory Treg cell, as shown using an A2Ar single knock-out mouse. Unexpectedly, when MC5r-/- post-EAU APC were used to activate A2Ar-/- post-EAU T cells the combination of cells significantly suppressed EAU, when transferred to EAU mice. In contrast, transfer of the reciprocal activation scheme did not suppress EAU. In order to explain this finding, MC5r-/-A2Ar-/- double knock-out (DKO) mice were bred. Naïve DKO mice had no differences in the APC populations, or inflammatory T cell subsets, but did have significantly more Treg cells. When we examined the number of CD4 and CD8 T cell subsets, we found significantly fewer CD8 T cells in the DKO mice compared to WT and both single knock-out mice. DKO mice also had significantly reduced EAU severity and accelerated resolution. In order to determine if the CD8 T cell deficiency contributed to the resistance to EAU in the DKO mice, we transferred naïve CD8 T cells from WT mice, that were immunized for EAU. Susceptibility to EAU was restored in DKO mice that received a CD8 T cell transfer. While the mechanism that contributed to the CD8 T cell deficiency in the DKO mice remains to be determined, these observations indicate an importance of CD8 T cells in the initiation of EAU. The involvement of CD4 and CD8 T cells suggests that both class I and class II antigen presentation can trigger an autoimmune response, suggesting a much wider range of antigens may trigger autoimmune disease.

scholarly journals Mitochondrial Dysfunction Associates With Acute T Lymphocytopenia and Impaired Functionality in COVID-19 Patients

2022 ◽  
Vol 12 ◽  
Author(s):  
Yufei Mo ◽  
Kelvin Kai-Wang To ◽  
Runhong Zhou ◽  
Li Liu ◽  
Tianyu Cao ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Mitochondrial Dysfunction ◽  
Cd8 T Cells ◽  
Functional Impairment ◽  
Cell Loss ◽  
Cd8 T Cell ◽  
Underlying Mechanism ◽  
T Cell Subsets ◽  
Effector Memory

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection results in rapid T lymphocytopenia and functional impairment of T cells. The underlying mechanism, however, remains incompletely understood. In this study, we focused on characterizing the phenotype and kinetics of T-cell subsets with mitochondrial dysfunction (MD) by multicolor flow cytometry and investigating the association between MD and T-cell functionality. While 73.9% of study subjects displayed clinical lymphocytopenia upon hospital admission, a significant reduction of CD4 or CD8 T-cell frequency was found in all asymptomatic, symptomatic, and convalescent cases. CD4 and CD8 T cells with increased MD were found in both asymptomatic and symptomatic patients within the first week of symptom onset. Lower proportion of memory CD8 T cell with MD was found in severe patients than in mild ones at the stage of disease progression. Critically, the frequency of T cells with MD in symptomatic patients was preferentially associated with CD4 T-cell loss and CD8 T-cell hyperactivation, respectively. Patients bearing effector memory CD4 and CD8 T cells with the phenotype of high MD exhibited poorer T-cell responses upon either phorbol 12-myristate-13-acetate (PMA)/ionomycin or SARS-CoV-2 peptide stimulation than those with low MD. Our findings demonstrated an MD-associated mechanism underlying SARS-CoV-2-induced T lymphocytopenia and functional impairment during the acute phase of infection.

scholarly journals Cortisol and epinephrine control opposing circadian rhythms in T cell subsets

Blood ◽  
2009 ◽  
Vol 113 (21) ◽  
pp. 5134-5143 ◽  
Author(s):  
Stoyan Dimitrov ◽  
Christian Benedict ◽  
Dennis Heutling ◽  
Jürgen Westermann ◽  
Jan Born ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Circadian Rhythms ◽  
Cd8 T Cells ◽  
Low Dose ◽  
Cd8 T Cell ◽  
T Cell Subsets ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Cell Counts

Abstract Pronounced circadian rhythms in numbers of circulating T cells reflect a systemic control of adaptive immunity whose mechanisms are obscure. Here, we show that circadian variations in T cell subpopulations in human blood are differentially regulated via release of cortisol and catecholamines. Within the CD4+ and CD8+ T cell subsets, naive cells show pronounced circadian rhythms with a daytime nadir, whereas (terminally differentiated) effector CD8+ T cell counts peak during daytime. Naive T cells were negatively correlated with cortisol rhythms, decreased after low-dose cortisol infusion, and showed highest expression of CXCR4, which was up-regulated by cortisol. Effector CD8+ T cells were positively correlated with epinephrine rhythms, increased after low-dose epinephrine infusion, and showed highest expression of β-adrenergic and fractalkine receptors (CX3CR1). Daytime increases in cortisol via CXCR4 probably act to redistribute naive T cells to bone marrow, whereas daytime increases in catecholamines via β-adrenoceptors and, possibly, a suppression of fractalkine signaling promote mobilization of effector CD8+ T cells from the marginal pool. Thus, activation of the major stress hormones during daytime favor immediate effector defense but diminish capabilities for initiating adaptive immune responses.

scholarly journals TCF-1 limits the formation of Tc17 cells via repression of the MAF–RORγt axis

2019 ◽  
Vol 216 (7) ◽  
pp. 1682-1699 ◽  
Author(s):  
Lisa A. Mielke ◽  
Yang Liao ◽  
Ella Bridie Clemens ◽  
Matthew A. Firth ◽  
Brigette Duckworth ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Fate ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Subsets ◽  
Double Positive ◽  
Cell Fate Decisions ◽  
Healthy Humans ◽  
Tc17 Cells

Interleukin (IL)-17–producing CD8+ T (Tc17) cells have emerged as key players in host-microbiota interactions, infection, and cancer. The factors that drive their development, in contrast to interferon (IFN)-γ–producing effector CD8+ T cells, are not clear. Here we demonstrate that the transcription factor TCF-1 (Tcf7) regulates CD8+ T cell fate decisions in double-positive (DP) thymocytes through the sequential suppression of MAF and RORγt, in parallel with TCF-1–driven modulation of chromatin state. Ablation of TCF-1 resulted in enhanced Tc17 cell development and exposed a gene set signature to drive tissue repair and lipid metabolism, which was distinct from other CD8+ T cell subsets. IL-17–producing CD8+ T cells isolated from healthy humans were also distinct from CD8+IL-17− T cells and enriched in pathways driven by MAF and RORγt. Overall, our study reveals how TCF-1 exerts central control of T cell differentiation in the thymus by normally repressing Tc17 differentiation and promoting an effector fate outcome.

scholarly journals Impact of multiple hits with cognate antigen on memory CD8+ T-cell fate

2020 ◽  
Vol 32 (9) ◽  
pp. 571-581 ◽  
Author(s):  
Shiki Takamura
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Memory T Cells ◽  
Cd8 T Cell ◽  
T Cell Subsets ◽  
Primary Immune Response ◽  
Memory T Cell ◽  
Cognate Antigen ◽  
Memory Cd8 T Cell

Abstract Antigen-driven activation of CD8+ T cells results in the development of a robust anti-pathogen response and ultimately leads to the establishment of long-lived memory T cells. During the primary response, CD8+ T cells interact multiple times with cognate antigen on distinct types of antigen-presenting cells. The timing, location and context of these antigen encounters significantly impact the differentiation programs initiated in the cells. Moderate re-activation in the periphery promotes the establishment of the tissue-resident memory T cells that serve as sentinels at the portal of pathogen entry. Under some circumstances, moderate re-activation of T cells in the periphery can result in the excessive expansion and accumulation of circulatory memory T cells, a process called memory inflation. In contrast, excessive re-activation stimuli generally impede conventional T-cell differentiation programs and can result in T-cell exhaustion. However, these conditions can also elicit a small population of exhausted T cells with a memory-like signature and self-renewal capability that are capable of responding to immunotherapy, and restoration of functional activity. Although it is clear that antigen re-encounter during the primary immune response has a significant impact on memory T-cell development, we still do not understand the molecular details that drive these fate decisions. Here, we review our understanding of how antigen encounters and re-activation events impact the array of memory CD8+ T-cell subsets subsequently generated. Identification of the molecular programs that drive memory T-cell generation will advance the development of new vaccine strategies that elicit high-quality CD8+ T-cell memory.

TNFR2 Is Expressed Preferentially By Late Differentiated CD8 T-Cells and Can be Triggered By TNFR2-Specific Ligand to Induce Cell Death of Recently Activated Antigen-Specific T Cells: A Possible Role of TNFR2 in T-Cell Deflation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4352-4352
Author(s):  
Mohammad Raeiszadeh ◽  
Matthew Verney ◽  
Charles Craddock ◽  
Harald Wajant ◽  
Paul Moss ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Receptor Expression ◽  
T Cell Subsets ◽  
Effector Memory ◽  
Cell Transplant ◽  
Gamma Chain ◽  
Transplant Patients

Abstract Recent evidence suggests that Tumor Necrosis Factor (TNF) can selectively kill antigen-specific autoreactive CD8+ T-cells through engagement with TNF Receptor 2 (TNFR2) (1). Within the immune system, TNFR2 expression is restricted to subsets of T-cells, a profile which is in marked contrast to the ubiquitous pattern of expression of TNFR1. However, the spectrum and physiological significance of TNFR2 expression by CD8+ T-cell subpopulations is unknown. In this study we analysed the expression of TNFR2 by CD8 T-cell subsets isolated from normal healthy donors by flow cytometry. In addition, in order to understand the physiological significance of TNFR2 expression on recently activated T cells, we further studied expression on CMV-specific CD8 T-cells which expanded in stem cell transplant patients in response to episodes of CMV reactivation. The expression of TNFR2 was compared to that of other common gamma chain receptors including IL2R and IL7R, and to the expression of a receptor for inflammatory cytokine IL6. TNFR2 expression was found to increase during differentiation of CD8+ T cells. In particular, TNFR2 expression was seen on 6.5% of naïve, 14.6% of central memory, 37.9% of effector memory and 45.2% of CD45RA-revertant effector memory (TEMRA) CD8+ T cells. In contrast, common gamma chain cytokine receptor expression was skewed towards less differentiated T-cell subsets. For example, IL-7R was expressed by 63% of central memory populations but only 18.4% of the TEMRA subset. Comparable expression of IL2R was 12.1% on TCM and 2% on TEMRA. Of interest, IL-6 receptor expression was predominantly expressed by naïve CD8 T-cells (69.5%). In support of these results, we went on to show that expression of TNFR2 was inducible on primary T cells following activation with anti-CD3 and IL-2 in vitro. Healthy CMV seropositive donors had a larger median number of CD8+ T cells expressing TNFR2 (53%) in comparison to CMV seronegative donors (15%), (p<0.0001), consistent with the known accumulation of differentiated T-cells within CMV seropositive individuals.The expression of TNFR2 was then examined on CMV-specific CD8 T-cells which were undergoing acute expansion in response to viremia in six haemopoietic stem cell transplant patients. The expansion of CMV-specific CD8 T-cells was accompanied by an increase in the intensity of TNFR2 expression which later decreased during the retraction of antigen-specific T-cells during resolution of viremia. In order to explore the functional significance of TNFR2 expression, T-cells isolated from healthy donors were treated with recombinant TNFR2-specific ligand. This induced cell loss ranging from 13% to 60% of all CD8 T-cells in relation to untreated control cells, with selective depletion of the TNFR2+ population. A similar proportion of CMV-specific T-cells from transplant patients were eliminated by ex vivo stimulation of TNFR2. In conclusion our work shows that TNFR2 expression increases during differentiation of CD8+ T cells. In addition, we were able to utilize virus-specific T cells from SCT patients to show that expression is increased during the acute response to stimulation with antigen. We also provide evidence that TNFR2 activation can lead to the partial elimination of antigen-specific CMV-specific T-cells and it may thus play an important role in the ‘deflation’ of a pathogen-specific T-cell immune response following resolution of infection. These data suggest that TNFR2 expression may act as a ligand to signal activation-induced cell death in late differentiated populations of CD8+ T cells. Further investigations are required to assess the molecular pathways of TNFR2 signalling that are activated following receptor ligation in vivoand whether or not these are disrupted in disorders associated with chronic CD8+ T cell lymphproliferation. (1) L. Ban et al, PNAS 2008, 105: 3644 Disclosures No relevant conflicts of interest to declare.

CD4 T lymphocytes are primed to express Fas ligand by CD4 cross-linking and to contribute to CD8 T-cell apoptosis via Fas/FasL death signaling pathway

Blood ◽  
2000 ◽  
Vol 96 (1) ◽  
pp. 195-202 ◽  
Author(s):  
Masaki Tateyama ◽  
Naoki Oyaizu ◽  
Thomas W. McCloskey ◽  
Soe Than ◽  
Savita Pahwa
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Interleukin 2 ◽  
Cd8 T Cell ◽  
T Cell Subsets ◽  
Cross Linking ◽  
Induced Apoptosis ◽  
Hiv 1

CD4 molecules serve as coreceptors for the T-cell receptor (TCR)/CD3 complex that are engaged coordinately with TCR and facilitate antigen-specific T-cell activation leading to interleukin 2 (IL-2) production and proliferation. However, cross-ligation of CD4 molecules prior to TCR stimulation has been shown to prime CD4 T cells to undergo apoptosis. Although in vivo and in vitro experiments have implicated the involvement of Fas/FasL interaction in this CD4 cross-linking (CD4XL)-induced apoptosis, detailed mechanisms to account for cell death induction have not been elucidated. In the present study, we demonstrate that CD4XL in purified T cells not only led to Fas up-regulation but also primed CD4 T cells to express FasL upon CD3 stimulation and rendered the T cells susceptible to Fas-mediated apoptosis. Notably, in addition to CD4+ T cells, CD4XL-induced sensitization for apoptosis was observed in CD8+ T cells as well and was associated with Bcl-x down-modulation. Both CD4 and CD8 T-cell subsets underwent apoptosis following cell–cell contact with FasL+ CD4 T cells. CD28 costimulation abrogated CD4XL/CD3-induced apoptosis with restoration of IL-2 production and prevented Bcl-x down-modulation. As CD4 molecules are the primary receptors for human immunodeficiency virus 1 (HIV-1), we conclude that HIV-1 envelope mediated CD4XL can lead to the generation of FasL-expressing CD4+ T cells that can lead to apoptosis of CD4 as well as CD8 T cells. These findings implicate a novel mechanism for CD8 T-cell depletion in HIV disease.

scholarly journals The architectural design of CD8+ T cell responses in acute and chronic infection: Parallel structures with divergent fates

2021 ◽  
Vol 218 (4) ◽  
Author(s):  
H. Kay Chung ◽  
Bryan McDonald ◽  
Susan M. Kaech
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Architectural Design ◽  
Viral Infections ◽  
Population Heterogeneity ◽  
T Cell Subsets ◽  
Dependent Manner ◽  
Cell Immunity ◽  
And Migration

In response to infection, T cells adopt a range of differentiation states, creating numerous heterogeneous subsets that exhibit different phenotypes, functions, and migration patterns. This T cell heterogeneity is a universal feature of T cell immunity, needed to effectively control pathogens in a context-dependent manner and generate long-lived immunity to those pathogens. Here, we review new insights into differentiation state dynamics and population heterogeneity of CD8+ T cells in acute and chronic viral infections and cancer and highlight the parallels and distinctions between acute and chronic antigen stimulation settings. We focus on transcriptional and epigenetic networks that modulate the plasticity and terminal differentiation of antigen-specific CD8+ T cells and generate functionally diverse T cell subsets with different roles to combat infection and cancer.

scholarly journals Naïve CD8 T cell IFNγ responses to a vacuolar antigen are regulated by an inflammasome-independent NLRP3 pathway and Toxoplasma gondii ROP5

2020 ◽  
Author(s):  
Angel K. Kongsomboonvech ◽  
Felipe Rodriguez ◽  
Anh L. Diep ◽  
Brandon M. Justice ◽  
Brayan E. Castallanos ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Toxoplasma Gondii ◽  
Cd8 T Cells ◽  
Immune Cell ◽  
Cd8 T Cell ◽  
Inflammasome Activation ◽  
Phenotypic Variance ◽  
Dependent Manner ◽  
Presentation Pathway

ABSTRACTHost resistance to Toxoplasma gondii relies on CD8 T cell IFNγ responses, which if modulated by the host or parasite could influence chronic infection and parasite transmission between hosts. Since host-parasite interactions that govern this response are not fully elucidated, we investigated requirements for eliciting naïve CD8 T cell IFNγ responses to a vacuolar resident antigen of T. gondii, TGD057. Naïve TGD057 antigen-specific CD8 T cells (T57) were isolated from transnuclear mice and responded to parasite-infected bone marrow-derived macrophages (BMDMs) in an antigen-dependent manner, first by producing IL-2 and then IFNγ. T57 IFNγ responses to TGD057 were independent of the parasite’s protein export machinery ASP5 and MYR1. Instead, host immunity pathways downstream of the regulatory Immunity-Related GTPases (IRG), including partial dependence on Guanylate-Binding Proteins, are required. Multiple T. gondii ROP5 isoforms and allele types, including ‘avirulent’ ROP5A from clade A and D parasite strains, were able to suppress CD8 T cell IFNγ responses to parasite-infected BMDMs. Phenotypic variance between clades B, C, D, F, and A strains suggest T57 IFNγ differentiation occurs independently of parasite virulence or any known IRG-ROP5 interaction. Consistent with this, removal of ROP5 is not enough to elicit maximal CD8 T cell IFNγ production to parasite-infected cells. Instead, macrophage expression of the pathogen sensors, NLRP3 and to a large extent NLRP1, were absolute requirements. Other members of the conventional inflammasome cascade are only partially required, as revealed by decreased but not abrogated T57 IFNγ responses to parasite-infected ASC, caspase-1/11, and gasdermin D deficient cells. Moreover, IFNγ production was only partially reduced in the absence of IL-12, IL-18 or IL-1R signaling. In summary, T. gondii effectors and host machinery that modulate parasitophorous vacuolar membranes, as well as NLR-dependent but inflammasome-independent pathways, determine the full commitment of CD8 T cells IFNγ responses to a vacuolar antigen.AUTHOR SUMMARYParasites are excellent “students” of our immune system as they can deflect, antagonize and confuse the immune response making it difficult to vaccinate against these pathogens. In this report, we analyzed how a widespread parasite of mammals, Toxoplasma gondii, manipulates an immune cell needed for immunity to many intracellular pathogens, the CD8 T cell. Host pathways that govern CD8 T cell production of the immune protective cytokine, IFNγ, were also explored. We hypothesized the secreted Toxoplasma virulence factor, ROP5, work to inhibit the MHC 1 antigen presentation pathway therefore making it difficult for CD8 T cells to see T. gondii antigens sequestered inside a parasitophorous vacuole. However, manipulation through T. gondii ROP5 does not fully explain how CD8 T cells commit to making IFNγ in response to infection. Importantly, CD8 T cell IFNγ responses to T. gondii require the pathogen sensor NLRP3 to be expressed in the infected cell. Other proteins associated with NLRP3 activation, including members of the conventional inflammasome activation cascade pathway, are only partially involved. Our results identify a novel pathway by which NLRP3 regulates T cell function and underscore the need for inflammasome-activating adjuvants in vaccines aimed at inducing CD8 T cell IFNγ responses to parasites.

scholarly journals Ecto-5′-Nucleotidase (CD73) Regulates the Survival of CD8+ T Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Mariana V. Rosemblatt ◽  
Brian Parra-Tello ◽  
Pedro Briceño ◽  
Elizabeth Rivas-Yáñez ◽  
Suat Tucer ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Interleukin 2 ◽  
Cd8 T Cell ◽  
Antigenic Stimulation ◽  
T Cell Subsets ◽  
Limited Information ◽  
Α Chain ◽  
The Impact

Ecto-5′-nucleotidase (CD73) is an enzyme present on the surface of tumor cells whose primary described function is the production of extracellular adenosine. Due to the immunosuppressive properties of adenosine, CD73 is being investigated as a target for new antitumor therapies. We and others have described that CD73 is present at the surface of different CD8+ T cell subsets. Nonetheless, there is limited information as to whether CD73 affects CD8+ T cell proliferation and survival. In this study, we assessed the impact of CD73 deficiency on CD8+ T cells by analyzing their proliferation and survival in antigenic and homeostatic conditions. Results obtained from adoptive transfer experiments demonstrate a paradoxical role of CD73. On one side, it favors the expression of interleukin-7 receptor α chain on CD8+ T cells and their homeostatic survival; on the other side, it reduces the survival of activated CD8+ T cells under antigenic stimulation. Also, upon in vitro antigenic stimulation, CD73 decreases the expression of interleukin-2 receptor α chain and the anti-apoptotic molecule Bcl-2, findings that may explain the reduced CD8+ T cell survival observed in this condition. These results indicate that CD73 has a dual effect on CD8+ T cells depending on whether they are subject to an antigenic or homeostatic stimulus, and thus, special attention should be given to these aspects when considering CD73 blockade in the design of novel antitumor therapies.

Peptide Vaccination Induces Dynamic Changes in CD4+ and CD8+ T Cell Subsets: Report on the First Peptide Vaccination Trial in Patients with Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3159-3159 ◽  
Author(s):  
Krzysztof Giannopoulos ◽  
Malgorzata Kowal ◽  
Anna Dmoszynska ◽  
Jacek Rolinski ◽  
Kamila Mazurek ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
T Cell Subsets ◽  
Peptide Vaccination ◽  
Tetramer Staining ◽  
Induced Changes

Abstract There is an accumulation of in vivo (graft-versus-leukemia effect) and in vitro (spontaneous remissions after infections) data providing evidence that CLL might be effectively targeted by T-cell based immunotherapy. Earlier, we characterized the receptor for hyaluronic acid mediated motility (RHAMM) as antigen associated with proliferation and negative prognosis in CLL. We also demonstrated that RHAMM-derived epitope(R3)- primed T cells were able to lyse RHAMM+ target CLL cells. Therefore, we initiated a small phase I/II clinical trial with R3 peptide vaccination for patients with CLL. Six CLL patients in Binet stage 0 of the disease were vaccinated four times at a biweekly interval with HLA-A2 restricted RHAMM-derived epitope R3 (ILSLELMKL, 300μg/dose on day 3) emulsified in incomplete Freund’s adjuvant (IFA) with concomitant administration of GM-CSF (100μg/dose, days 1–5). R3-specific T-cell responses were assessed by tetramer staining and ELISPOT assays. T-cell subsets which play a role in regulation of immune responses including CD3+CD4+CD25hiCD127loFOXP3+ T regs, Th17, CD8+CD137+, CD8+CD103+ and IL-17 producing CD8+ T cells (CD8+IL-17+) were evaluated by flow cytometry. No severe adverse events greater than CTC Io skin toxicity could be observed. Four of six patients showed a reduction of WBC during vaccination. Although these WBC changes did not meet the NCI response criteria, we described these favorable hematological changes achieved in short period of immunotherapy as hematological improvement (defined as at least 20% reduction of WBC during vaccination). The immune responses were found in 5/6 patients as assessed by tetramer-staining (positive response defined as an increase of R3-specific CD8+ T cell frequency by more than 100% after vaccination) and confirmed in 4/5 as assessed by ELISPOT assay. Patients included in this study showed median Tregs frequency of 4.2%, range: 2.5–8%. There was no significant difference of Tregs percentages between patients who improved clinically when compared with non-responders (median 6.1% vs. 3.7%). Vaccination induced Tregs in 4 patients (2 non-responders and 2 responders). Two other patients who improved hematologically did not significantly change frequency of Tregs or even reduced it during vaccination (Figure 1). Median expression of CD103 on CD8+ T cells was 1.84%, range: 0.41–5.63%. In one non-responder, we observed an increase in frequency of CD103+CD8+ T-cells during vaccination from 1.46% to 2.56%. During vaccination, changes in CD8+CD103+ T cell subset did not correlate with the frequency of Tregs, nonetheless we could find an inverse correlation with inflammatory Th17 T cells (r2=−0.5, p&lt;0.05). We could find a correlation between the frequency of Tregs and activated CD8+CD69+ T cells (r2=0.51, p&lt;0.05). Interestingly, CD8+CD137+ cells correlated with CD8+IL-17+ T cells (r2=0.54, p&lt;0.05). In conclusion, peptide vaccination in CLL patients is safe and feasible to mount immune responses against the tumor antigen RHAMM. Most of patients benefited hematologically from vaccination. Although in some patients we observed an induction of tumor-specific T cells without induction of Tregs there is a rationale to add novel active agents against Tregs in future vaccination trials. Figure 1. Peptide vaccination induced changes in WBC, percentages of regulatory T cells (Tregs) as well as R3 specific tetramer ‘CD’ T cells (tetra) of A CLL patients. Patients B, C, E and F improved hematologically during vaccination. Figure 1. Peptide vaccination induced changes in WBC, percentages of regulatory T cells (Tregs) as well as R3 specific tetramer ‘CD’ T cells (tetra) of A CLL patients. Patients B, C, E and F improved hematologically during vaccination.

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