scholarly journals Peripheral self-reactivity regulates antigen-specific CD8 T-cell responses and cell division under physiological conditions

Open Biology ◽  
2016 ◽  
Vol 6 (11) ◽  
pp. 160293 ◽  
Author(s):  
Lee Kim Swee ◽  
Zhen Wei Tan ◽  
Anna Sanecka ◽  
Nagisa Yoshida ◽  
Harshil Patel ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Toxoplasma Gondii ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Effector Function ◽  
Effector Functions ◽  
Physiological Conditions ◽  
Cell Clones

T-cell identity is established by the expression of a clonotypic T-cell receptor (TCR), generated by somatic rearrangement of TCRα and β genes. The properties of the TCR determine both the degree of self-reactivity and the repertoire of antigens that can be recognized. For CD8 T cells, the relationship between TCR identity—hence reactivity to self—and effector function(s) remains to be fully understood and has rarely been explored outside of the H-2 b haplotype. We measured the affinity of three structurally distinct CD8 T-cell-derived TCRs that recognize the identical H-2 L d -restricted epitope, derived from the Rop7 protein of Toxoplasma gondii . We used CD8 T cells obtained from mice generated by somatic cell nuclear transfer as the closest approximation of primary T cells with physiological TCR rearrangements and TCR expression levels. First, we demonstrate the common occurrence of secondary rearrangements in endogenously rearranged loci. Furthermore, we characterized and compared the response of Rop7-specific CD8 T-cell clones upon Toxoplasma gondii infection as well as effector function and TCR signalling upon antigenic stimulation in vitro . Antigen-independent TCR cross-linking in vitro uncovered profound intrinsic differences in the effector functions between T-cell clones. Finally, by assessing the degree of self-reactivity and comparing the transcriptomes of naive Rop7 CD8 T cells, we show that lower self-reactivity correlates with lower effector capacity, whereas higher self-reactivity is associated with enhanced effector function as well as cell cycle entry under physiological conditions. Altogether, our data show that potential effector functions and basal proliferation of CD8 T cells are set by self-reactivity thresholds.

scholarly journals DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

2008 ◽  
Vol 205 (13) ◽  
pp. 2965-2973 ◽  
Author(s):  
Susan Gilfillan ◽  
Christopher J. Chan ◽  
Marina Cella ◽  
Nicole M. Haynes ◽  
Aaron S. Rapaport ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Adhesion Molecules ◽  
Cd8 T Cells ◽  
Nk Cell ◽  
Cd8 T Cell ◽  
Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

scholarly journals A new in vitro model of polymyositis reveals CD8+ T cell invasion into muscle cells and its cytotoxic role

Rheumatology ◽  
2019 ◽  
Vol 59 (1) ◽  
pp. 224-232
Author(s):  
Mari Kamiya ◽  
Fumitaka Mizoguchi ◽  
Akito Takamura ◽  
Naoki Kimura ◽  
Kimito Kawahata ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Muscle Injury ◽  
In Vitro Model ◽  
Muscle Cells ◽  
Cd8 T Cell ◽  
Class I ◽  
Vitro Model

Abstract Objectives The hallmark histopathology of PM is the presence of CD8+ T cells in the non-necrotic muscle cells. The aim of this study was to clarify the pathological significance of CD8+ T cells in muscle cells. Methods C2C12 cells were transduced retrovirally with the genes encoding MHC class I (H2Kb) and SIINFEKL peptide derived from ovalbumin (OVA), and then differentiated to myotubes (H2KbOVA-myotubes). H2KbOVA-myotubes were co-cultured with OT-I CD8+ T cells derived from OVA-specific class I restricted T cell receptor transgenic mice as an in vitro model of PM to examine whether the CD8+ T cells invade into the myotubes and if the myotubes with the invasion are more prone to die than those without. Muscle biopsy samples from patients with PM were examined for the presence of CD8+ T cells in muscle cells. The clinical profiles were compared between the patients with and without CD8+ T cells in muscle cells. Results Analysis of the in vitro model of PM with confocal microscopy demonstrated the invasion of OT-I CD8+ T cells into H2KbOVA-myotubes. Transmission electron microscopic analysis revealed an electron-lucent area between the invaded CD8+ T cell and the cytoplasm of H2KbOVA-myotubes. The myotubes invaded with OT-I CD8+ T cells died earlier than the uninvaded myotubes. The level of serum creatinine kinase was higher in patients with CD8+ T cells in muscle cells than those without these cells. Conclusion CD8+ T cells invade into muscle cells and contribute to muscle injury in PM. Our in vitro model of PM is useful to examine the mechanisms underlying muscle injury induced by CD8+ T cells.

BK Polyomavirus–Specific CD8 T-Cell Expansion In Vitro Using 27mer Peptide Antigens for Developing Adoptive T-Cell Transfer and Vaccination

2020 ◽  
Author(s):  
Maud Wilhelm ◽  
Amandeep Kaur ◽  
Marion Wernli ◽  
Hans H Hirsch
Keyword(s):  
T Cells ◽  
T Cell ◽  
Kidney Transplant ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
T Cell Responses ◽  
Cd8 T Cell ◽  
Cell Responses ◽  
Bk Polyomavirus

Abstract Background BK polyomavirus (BKPyV) remains a significant cause of premature kidney transplant failure. In the absence of effective antivirals, current treatments rely on reducing immunosuppression to regain immune control over BKPyV replication. Increasing BKPyV-specific CD8 T cells correlate with clearance of BKPyV DNAemia in kidney transplant patients. We characterized a novel approach for expanding BKPyV-specific CD8 T cells in vitro using 27mer-long synthetic BKPyV peptides, different types of antigen-presenting cells, and CD4 T cells. Methods Langerhans cells and immature or mature monocyte-derived dendritic cells (Mo-DCs) were generated from peripheral blood mononuclear cells of healthy blood donors, pulsed with synthetic peptide pools consisting of 36 overlapping 27mers (27mP) or 180 15mers (15mP). BKPyV-specific CD8 T-cell responses were assessed by cytokine release assays using 15mP or immunodominant 9mers. Results BKPyV-specific CD8 T cells expanded using 27mP and required mature Mo-DCs (P = .0312) and CD4 T cells (P = .0156) for highest responses. The resulting BKPyV-specific CD8 T cells proliferated, secreted multiple cytokines including interferon γ and tumor necrosis factor α, and were functional (CD107a+/PD1–) and cytotoxic. Conclusions Synthetic 27mP permit expanding BKPyV-specific CD8 T-cell responses when pulsing mature Mo-DCs in presence of CD4 T cells, suggesting novel and safe approaches to vaccination and adoptive T-cell therapies for patients before and after kidney transplantation.

scholarly journals IL4I1 Accelerates the Expansion of Effector CD8+ T Cells at the Expense of Memory Precursors by Increasing the Threshold of T-Cell Activation

2020 ◽  
Vol 11 ◽  
Author(s):  
Marie-Line Puiffe ◽  
Aurélie Dupont ◽  
Nouhoum Sako ◽  
Jérôme Gatineau ◽  
José L. Cohen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Activation ◽  
Cell Clone ◽  
Cd8 T Cell ◽  
Lymphocytic Choriomeningitis ◽  
T Cell Clone

IL4I1 is an immunoregulatory enzyme that inhibits CD8 T-cell proliferation in vitro and in the tumoral context. Here, we dissected the effect of IL4I1 on CD8 T-cell priming by studying the differentiation of a transgenic CD8 T-cell clone and the endogenous repertoire in a mouse model of acute lymphocytic choriomeningitis virus (LCMV) infection. Unexpectedly, we show that IL4I1 accelerates the expansion of functional effector CD8 T cells during the first several days after infection and increases the average affinity of the elicited repertoire, supporting more efficient LCMV clearance in WT mice than IL4I1-deficient mice. Conversely, IL4I1 restrains the differentiation of CD8 T-cells into long-lived memory precursors and favors the memory response to the most immunodominant peptides. IL4I1 expression does not affect the phenotype or antigen-presenting functions of dendritic cells (DCs), but directly reduces the stability of T-DC immune synapses in vitro, thus dampening T-cell activation. Overall, our results support a model in which IL4I1 increases the threshold of T-cell activation, indirectly promoting the priming of high-affinity clones while limiting memory T-cell differentiation.

scholarly journals Priming of Antiviral CD8 T Cells without Effector Function by a Persistently Replicating Hepatitis C-Like Virus

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
Alex S. Hartlage ◽  
Christopher M. Walker ◽  
Amit Kapoor
Keyword(s):  
T Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
T Cell ◽  
Animal Models ◽  
Cd8 T Cells ◽  
Persistent Infection ◽  
Cd8 T Cell ◽  
Effector Function ◽  
Viral Escape

ABSTRACT Immune-competent animal models for the hepatitis C virus (HCV) are nonexistent, impeding studies of host-virus interactions and vaccine development. Experimental infection of laboratory rats with a rodent hepacivirus isolated from Rattus norvegicus (RHV) is a promising surrogate model due to its recapitulation of HCV-like chronicity. However, several aspects of rat RHV infection remain unclear, for instance, how RHV evades host adaptive immunity to establish persistent infection. Here, we analyzed the induction, differentiation, and functionality of RHV-specific CD8 T cell responses that are essential for protection against viral persistence. Virus-specific CD8 T cells targeting dominant and subdominant major histocompatibility complex class I epitopes proliferated considerably in liver after RHV infection. These populations endured long term yet never acquired antiviral effector functions or selected for viral escape mutations. This was accompanied by the persistent upregulation of programmed cell death-1 and absent memory cell formation, consistent with a dysfunctional phenotype. Remarkably, transient suppression of RHV viremia with a direct-acting antiviral led to the priming of CD8 T cells with partial effector function, driving the selection of a viral escape variant. These data demonstrate an intrinsic abnormality within CD8 T cells primed by rat RHV infection, an effect that is governed at least partially by the magnitude of early virus replication. Thus, this model could be useful in investigating mechanisms of CD8 T cell subversion, leading to the persistence of hepatotropic pathogens such as HCV. IMPORTANCE Development of vaccines against hepatitis C virus (HCV), a major cause of cirrhosis and cancer, has been stymied by a lack of animal models. The recent discovery of an HCV-like rodent hepacivirus (RHV) enabled the development of such a model in rats. This platform recapitulates HCV hepatotropism and viral chronicity necessary for vaccine testing. Currently, there are few descriptions of RHV-specific responses and why they fail to prevent persistent infection in this model. Here, we show that RHV-specific CD8 T cells, while induced early at high magnitude, do not develop into functional effectors capable of controlling virus. This defect was partially alleviated by short-term treatment with an HCV antiviral. Thus, like HCV, RHV triggers dysfunction of virus-specific CD8 T cells that are vital for infection resolution. Additional study of this evasion strategy and how to mitigate it could enhance our understanding of hepatotropic viral infections and lead to improved vaccines and therapeutics.

scholarly journals Phenotypic and Functional Separation of Memory and Effector Human CD8+ T Cells

1997 ◽  
Vol 186 (9) ◽  
pp. 1407-1418 ◽  
Author(s):  
Dörte Hamann ◽  
Paul A. Baars ◽  
Martin H.G. Rep ◽  
Berend Hooibrink ◽  
Susana R. Kerkhof-Garde ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Fas Ligand ◽  
Cytotoxic T Lymphocyte ◽  
Cell Subset ◽  
Cd8 T Cell ◽  
Cytolytic Activity ◽  
T Cell Subset

Human CD8+ memory- and effector-type T cells are poorly defined. We show here that, next to a naive compartment, two discrete primed subpopulations can be found within the circulating human CD8+ T cell subset. First, CD45RA−CD45R0+ cells are reminiscent of memory-type T cells in that they express elevated levels of CD95 (Fas) and the integrin family members CD11a, CD18, CD29, CD49d, and CD49e, compared to naive CD8+ T cells, and are able to secrete not only interleukin (IL) 2 but also interferon γ, tumor necrosis factor α, and IL-4. This subset does not exert cytolytic activity without prior in vitro stimulation but does contain virus-specific cytotoxic T lymphocyte (CTL) precursors. A second primed population is characterized by CD45RA expression with concomitant absence of expression of the costimulatory molecules CD27 and CD28. The CD8+CD45RA+CD27− population contains T cells expressing high levels of CD11a, CD11b, CD18, and CD49d, whereas CD62L (L-selectin) is not expressed. These T cells do not secrete IL-2 or -4 but can produce IFN-γ and TNF-α. In accordance with this finding, cells contained within this subpopulation depend for proliferation on exogenous growth factors such as IL-2 and -15. Interestingly, CD8+CD45RA+CD27− cells parallel effector CTLs, as they abundantly express Fas-ligand mRNA, contain perforin and granzyme B, and have high cytolytic activity without in vitro prestimulation. Based on both phenotypic and functional properties, we conclude that memory- and effector-type T cells can be separated as distinct entities within the human CD8+ T cell subset.

scholarly journals Human memory CD8 T cell effector potential is epigenetically preserved during in vivo homeostasis

2017 ◽  
Vol 214 (6) ◽  
pp. 1593-1606 ◽  
Author(s):  
Hossam A. Abdelsamed ◽  
Ardiana Moustaki ◽  
Yiping Fan ◽  
Pranay Dogra ◽  
Hazem E. Ghoneim ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Human Memory ◽  
Memory Cd8 T Cells ◽  
Memory Cd8 T Cell ◽  
Cell Effector

Antigen-independent homeostasis of memory CD8 T cells is vital for sustaining long-lived T cell–mediated immunity. In this study, we report that maintenance of human memory CD8 T cell effector potential during in vitro and in vivo homeostatic proliferation is coupled to preservation of acquired DNA methylation programs. Whole-genome bisulfite sequencing of primary human naive, short-lived effector memory (TEM), and longer-lived central memory (TCM) and stem cell memory (TSCM) CD8 T cells identified effector molecules with demethylated promoters and poised for expression. Effector-loci demethylation was heritably preserved during IL-7– and IL-15–mediated in vitro cell proliferation. Conversely, cytokine-driven proliferation of TCM and TSCM memory cells resulted in phenotypic conversion into TEM cells and was coupled to increased methylation of the CCR7 and Tcf7 loci. Furthermore, haploidentical donor memory CD8 T cells undergoing in vivo proliferation in lymphodepleted recipients also maintained their effector-associated demethylated status but acquired TEM-associated programs. These data demonstrate that effector-associated epigenetic programs are preserved during cytokine-driven subset interconversion of human memory CD8 T cells.

Development of a Nonhuman Primate Model for Analysis of the Adoptive Transfer of Antigen-Specific T Cell Clones.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 770-770
Author(s):  
Carolina Berger ◽  
Michael Jensen ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Adoptive Transfer ◽  
Cell Transfer ◽  
Primate Model ◽  
T Cell Clones ◽  
Cell Clones

Abstract In principle, the adoptive transfer of T cell clones specific for antigens expressed by pathogens or malignant cells could be therapeutically effective and allow precise control of the specificity, function, and magnitude of T cell immunity. However, the infusion of large numbers of cultured T cells or T cell clones in clinical trials has frequently failed to eradicate tumors or provide long-term control of infection. This may be due in part to the acquisition of an effector phenotype by the T cells during in vitro culture, which reduces their ability to survive in vivo and establish an immune response of sufficient magnitude for sustained efficacy. Several approaches including the administration of cytokines such as IL15, or lymphodepletion prior to cell transfer might promote the establishment of T cell memory after T cell transfer. To facilitate the rational development of clinical trials of T cell therapy, we have employed a nonhuman primate model of adoptive T cell transfer in which culture conditions and cell doses identical to those in human studies are utilized, and designed strategies to permit rigorous analysis of the persistence, function, phenotype, and migration of transferred cells. CD8+ CTL specific for macaque CMV were detected using an overlapping peptide panel and cytokine flow cytometry, isolated as individual T cell clones by limiting dilution, and propagated to large numbers in vitro. The T cell clones were transduced to express an intracellular truncated CD19 (ΔCD19) surface marker to allow tracking and functional assessment of T cells in vivo, and enriched by immunomagnetic selection to high purity (>98%) prior to transfer. The persistence of transferred ΔCD19+ T cells in the blood and their migration to the bone marrow and lymph nodes was determined by flow cytometry after staining with anti CD19, CD8, and CD3 antibodies. The infusion of ΔCD19+CD8+ CTL (3 x 108/kg) was safe and the cells remained detectable in vivo for >5 months. ΔCD19+CD8+ T cells were easily detected in the blood 1 day after transfer at a level of 2.7% of CD8+ T cells and gradually declined over 56 days to a stable population of 0.15–0.2% of CD8+ T cells. At the time of transfer the ΔCD19+CD8+ T cells had an effector phenotype (CD62L− CD127−), but gradually converted to a CD62L+CD127+ memory phenotype in vivo. The infused T cells were found at high levels in lymph node and bone marrow at day 14 after transfer (1.4% and 2.5%, respectively) and the cells at these sites were predominantly CD62L+. The ΔCD19+CD62L+ T cells lacked direct lytic function and expressed low levels of granzyme B, consistent with memory T cells. Sorting of these cells from post-transfer PBMC showed that in vitro activation restored lytic activity. The transferred ΔCD19+CD62L+ T cells in post-infusion PBMC produced IFNγ and TNFα comparable to endogenous CMV-specific CD8+ CTL. These results demonstrate that a subset (5–10%) of transferred CD8+ CTL clones can persist long-term as functional memory T cells. The macaque CD8+ T cell clones are responsive to IL15 in vitro and a safe regimen for administering IL15 to macaques that boosts endogenous T cells has been identified. Studies are now in progress to determine if IL15 can enhance the efficiency with which effector and memory CD8+ T cell responses can be augmented after adoptive transfer of T cell clones.

AAV-2 Capsid-Specific CD8+ T Cells Limit the Duration of Gene Therapy in Humans and Cross-React with AAV-8 Capsid.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 455-455 ◽  
Author(s):  
Federico Mingozzi ◽  
Marcela V. Maus ◽  
Denise E. Sabatino ◽  
Daniel J. Hui ◽  
John E.J. Rasko ◽  
...  
Keyword(s):  
Gene Therapy ◽  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Transgene Expression ◽  
Cd8 T Cell ◽  
Target Cells

Abstract Efforts to establish an adeno-associated viral (AAV) vector-mediated gene therapy for the treatment of hemophilia B have been hindered by an immune response to the viral capsid antigen. Preclinical studies in small and large animal models of the disease showed long-term factor IX (F.IX) transgene expression and correction of the phenotype. However, in a recent phase I/II clinical trial in humans (Manno et al., Nat. Med. 2006), after hepatic gene transfer with an AAV-2 vector expressing human F.IX transgene, expression lasted for only a few weeks, declining to baseline concurrently with a peak in liver enzymes. We hypothesized that T cells directed towards AAV capsid antigens displayed by transduced hepatocytes were activated and these mediated destruction of the transduced hepatocytes, thereby causing loss of transgene expression and a transient transaminitis. Peripheral blood mononuclear cells isolated from AAV-infused subjects were stained with an AAV capsid-specific MHC class I pentamer either directly or after in vitro expansion. Two weeks after vector infusion 0.14% of circulating CD8+ T cells were capsid-specific on direct staining, and five weeks after infusion the capsid-specific population had expanded to 0.5% of the circulating CD8+ T cells, indicating proliferation of this T cell subset. By 20 weeks after vector infusion, the capsid-specific CD8+ T cell population had contracted to the level seen at 2 weeks. The expansion and contraction of this capsid-specific CD8+ T cell population paralleled the rise and fall of serum transaminases in the subject observed. Subsequent ex vivo studies of PBMC showed the presence of a readily expandable pool of capsid-specific CD8+ T cells up to 2.5 years post vector-infusion. Similarly, we were able to expand AAV-specific CD8+ T cells from peripheral blood of normal donors, suggesting the existence of a T cell memory pool. Expanded CD8+ T cells were functional as evidenced by specific lysis of HLA-matched target cells and by IFN-γsecretion in response to AAV epitopes. It has been argued that potentially harmful immune responses could be avoided by switching AAV serotypes, however, capsid protein sequences are highly conserved among different serotypes, as are some immunodominant epitopes that we identified. Indeed, we demonstrated that capsid-specific CD8+ T cells from AAV-infused hemophilic subjects functionally cross-react with AAV-8. Moreover, cells expanded from normal donors with AAV-2 vector capsids proliferated upon culture with AAV-8 capsids, demonstrating that both vectors could be processed appropriately in vitro to present the epitopic peptide to capsid-specific T cells. This suggests that AAV-2-specific memory CD8+ T cells normally present in humans likely would expand upon exposure to AAV-8 capsid epitopes. We conclude that the use of immunomodulatory therapy may be a better approach to achieving durable transgene expression in the setting of AAV-mediated gene therapy.

ROR1 Specific T Cell Clones from Healthy Individuals Show Common T Cell Receptor Motifs

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3364-3364
Author(s):  
Falk Heidenreich ◽  
Elke Ruecker-Braun ◽  
Juliane S. Stickel ◽  
Anne Eugster ◽  
Denise Kühn ◽  
...  
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
T Cell ◽  
Mhc Class I ◽  
T Cell Receptor ◽  
Cd8 T Cells ◽  
Cd8 T Cell ◽  
Cell Receptor ◽  
T Cell Clones ◽  
Cell Clones

Abstract Background Immunotherapy for CLL with new antibodies or T-cells with modified TCR relies on attractive targets. ROR1 is such a promising target since it is highly overexpressed in CLL. Chimeric antigen receptor engineered T cells and antibodies directed against the extracellular part of ROR1 have already been developed and tested in vitro or in animal models, but still there is no MHC-class I presented peptide serving as target structure for CD8+ T cells (with or without a genetically modified T cell receptor) available. Aim The aim of this study was (1) to identify an immunogenic MHC-class I presented ROR1 peptide, (2) to generate respective ROR1 peptide specific CD8+ T cell clones, and (3) to analyze the nucleotide sequence of the CDR3 region of the expressed alpha and beta T cell receptor chain. Results In mass spectrometric-based analyses of the HLA-ligandome a HLA-B*07 presented ROR1 peptide was identified in primary CLL cells of two patients. Six T cell clones specific for this particular ROR1-peptide were generated from single CD8+ T cells from 2 healthy individuals with 3 T cell clones generated from each donor. Functionality and specificity of those T cell clones were tested in cytotoxicity assays. All 6 dextramer+ CD8+ T cell clones lysed peptide loaded and HLA-B*07+ transduced K562 cells (kindly provided by Lorenz Jahn, [Jahn et al., Blood, 2015 Feb 5;125(6):949-58]). Two selected clones (XD8 and XB6) were tested for their cytotoxic potential against 2 ROR1+ HLA-B*07+ tumor cell lines (with the ROR1 peptide identified by mass spectrometry for both of them) and against 2 primary CLL cell samples. Tested clones showed a significant lysis of the respective target cells. CDR3 regions of the alpha and beta T cell receptor chain were sequenced on a single cell level. The CDR3 alpha region from each of the 3 ROR1 specific T cell clones from donor A showed some similarities to T cell clones derived from donor B (Table 1). Conclusion For the first time a MHC-class I presented ROR1 peptide antigen is reported. ROR1 positive CLL cells can be targeted by specific HLA-B*07 restricted CTLs. Respective CD8+ T cell clones with anti-leukemic activity from 2 donors share some amino acid motifs of the CDR3 alpha and beta regions. In conclusion, this information provides the possibility of generating ROR1 specific CD8+ T cells with genetically modified T cell receptors for immunotherapy and for tracking those cells after administration with next generation sequencing in peripheral blood samples of patients. Furthermore, data suggest the existence of public TCR motifs in leukemia antigen specific CTLs, which needs to be proven in follow-up experiments with larger cohorts of donors and patients. Finally, the presented strategy to identify leukemia specific peptide antigens for CD8+ T cells might be an attractive method for similar projects. Table 1 Amino acid sequences of CDR3 alpha and beta regions of the TCR of ROR1 specific CD8+ T cell clones. When comparing two clones, matching amino acids are depicted in red. The aromatic amino acids phenylalanine (F) and tyrosine (Y) are shown in blue when situated at the same position. Gaps inserted during the sequence alignment process are indicated by a hyphen '-'. Table 1. Amino acid sequences of CDR3 alpha and beta regions of the TCR of ROR1 specific CD8+ T cell clones. When comparing two clones, matching amino acids are depicted in red. The aromatic amino acids phenylalanine (F) and tyrosine (Y) are shown in blue when situated at the same position. Gaps inserted during the sequence alignment process are indicated by a hyphen '-'. Disclosures Middeke: Sanofi: Honoraria. Schetelig:Sanofi: Honoraria.

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