scholarly journals Generation of antigen receptor-specific suppressor T cell clones in man.

1986 ◽  
Vol 164 (3) ◽  
pp. 950-955 ◽  
Author(s):  
N Mohagheghpour ◽  
N K Damle ◽  
S Takada ◽  
E G Engleman
Keyword(s):  
T Cells ◽  
T Cell ◽  
Antigen Receptor ◽  
Suppressor Cells ◽  
Suppressive Effect ◽  
T Cell Clones ◽  
Suppressor T Cell ◽  
Hla Dr ◽  
Cell Clones

We have shown previously that CD8+ T cells proliferate upon exposure to autologous, antigen primed CD4+ T cells, and suppress the response of fresh T cells to the priming antigen but not irrelevant antigens. The stimulus and target of suppression in this system appears to be the antigen receptor on the surface of CD4+ cells, rather than the nominal antigen. In the current study, alloantigen primed CD4+ inducer cells and IL-2-containing medium were used to generate clones of suppressor cells from several individuals. The clones inhibited the response of fresh autologous T cells only to the original allogeneic stimulator cell and to stimulator cells that shared HLA-DR antigens with the priming cell. The clones were also genetically restricted, since they inhibited the response of HLA-A,B-compatible but not HLA-A,B-incompatible individuals. The availability of a method for reproducibly generating antigen receptor-specific suppressor T cell clones in vitro should make it possible to clarify the mechanism, whereby such cells are activated and exert their suppressive effect.

scholarly journals An Ex Vivo Study of T Lymphocytes Recovered from the Lungs of I/St Mice Infected with and Susceptible toMycobacterium tuberculosis

1998 ◽  
Vol 66 (10) ◽  
pp. 4981-4988 ◽  
Author(s):  
Irina Lyadova ◽  
Vladimir Yeremeev ◽  
Konstantin Majorov ◽  
Boris Nikonenko ◽  
Sergei Khaidukov ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Ex Vivo ◽  
Antimycobacterial Activity ◽  
Enzymatic Digestion ◽  
Interleukin 4 ◽  
T Cell Clones ◽  
Cell Clones ◽  
Ifn Γ

ABSTRACT I/St mice, previously characterized as susceptible toMycobacterium tuberculosis H37Rv, were given 103 or 105 CFU intravenously. At two time points postinoculation, the cell suspensions that resulted from enzymatic digestion of lungs were enumerated and further characterized phenotypically and functionally. Regarding the T-cell populations recovered at 2 and 5 weeks postinfection, two main results were obtained: (i) the population of CD44− CD45RB+cells disappeared within 2 weeks postinfection, while the number of CD44+ CD45RB−/low cells slowly increased between weeks 2 and 5; (ii) when cocultured with irradiated syngeneic splenocytes, these lung T cells proliferated in the presence of H37Rv sonicate. Using H37Rv sonicate and irradiated syngeneic splenocytes to reactivate lung T cells, we selected five CD3+CD4+ CD8− T-cell clones. In addition to the H37Rv sonicate, the five clones react to both a short-term culture filtrate and an affinity-purified 15- to 18-kDa mycobacterial molecule as assessed by the proliferative assay. However, there was a clear difference between T-cell clones with respect to cytokine (gamma interferon [IFN-γ] and interleukin-4 [IL-4] and IL-10) profiles: besides one Th1-like (IFN-γ+ IL-4−) clone and one Th0-like (IFN-γ+ IL-4+IL-10+) clone, three clones produced predominantly IL-10, with only marginal or no IL-4 and IFN-γ responses. Inhibition of mycobacterial growth by macrophages in the presence of T cells was studied in a coculture in vitro system. It was found that the capacity to enhance antimycobacterial activity of macrophages fully correlated with INF-γ production by individual T-cell clones following genetically restricted recognition of infected macrophages. The possible functional significance of cytokine diversity among T-cell clones is discussed.

scholarly journals African Green Monkey TRIM5α Restriction in Simian Immunodeficiency Virus-Specific Rhesus Macaque Effector CD4 T Cells Enhances Their Survival and Antiviral Function

2015 ◽  
Vol 89 (8) ◽  
pp. 4449-4456 ◽  
Author(s):  
Sumiti Jain ◽  
Matthew T. Trivett ◽  
Victor I. Ayala ◽  
Claes Ohlen ◽  
David E. Ott
Keyword(s):  
T Cells ◽  
T Cell ◽  
Rhesus Macaque ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
T Cell Clones ◽  
Cell Clones ◽  
Immunodeficiency Virus ◽  
Antiviral Function

ABSTRACTThe expression of xenogeneic TRIM5α proteins can restrict infection in various retrovirus/host cell pairings. Previously, we have shown that African green monkey TRIM5α (AgmTRIM5α) potently restricts both human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus mac239 (SIVmac239) replication in a transformed human T-cell line (L. V. Coren, et al., Retrovirology 12:11, 2015,http://dx.doi.org/10.1186/s12977-015-0137-9). To assess AgmTRIM5α restriction in primary cells, we transduced AgmTRIM5α into primary rhesus macaque CD4 T cells and infected them with SIVmac239. Experiments with T-cell clones revealed that AgmTRIM5α could reproducibly restrict SIVmac239replication, and that this restriction synergizes with an intrinsic resistance to infection present in some CD4 T-cell clones. AgmTRIM5α transduction of virus-specific CD4 T-cell clones increased and prolonged their ability to suppress SIV spread in CD4 target cells. This increased antiviral function was strongly linked to decreased viral replication in the AgmTRIM5α-expressing effectors, consistent with restriction preventing the virus-induced cytopathogenicity that disables effector function. Taken together, our data show that AgmTRIM5α restriction, although not absolute, reduces SIV replication in primary rhesus CD4 T cells which, in turn, increases their antiviral function. These results support priorin vivodata indicating that the contribution of virus-specific CD4 T-cell effectors to viral control is limited due to infection.IMPORTANCEThe potential of effector CD4 T cells to immunologically modulate SIV/HIV infection likely is limited by their susceptibility to infection and subsequent inactivation or elimination. Here, we show that AgmTRIM5α expression inhibits SIV spread in primary effector CD4 T cellsin vitro. Importantly, protection of effector CD4 T cells by AgmTRIM5α markedly enhanced their antiviral function by delaying SIV infection, thereby extending their viability despite the presence of virus. Ourin vitrodata support priorin vivoHIV-1 studies suggesting that the antiviral CD4 effector response is impaired due to infection and subsequent cytopathogenicity. The ability of AgmTRIM5α expression to restrict SIV infection in primary rhesus effector CD4 T cells now opens an opportunity to use the SIV/rhesus macaque model to further elucidate the potential and scope of anti-AIDS virus effector CD4 T-cell function.

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.

Establishing T Cell Memory by Adoptive Transfer of T Cell Clones.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 866-866
Author(s):  
Carolina Berger ◽  
Michael C. Jensen ◽  
Stanley R. Riddell
Keyword(s):  
T Cells ◽  
T Cell ◽  
Adoptive Transfer ◽  
Annexin V ◽  
T Cell Memory ◽  
T Cell Clones ◽  
Cell Clones

Abstract Adoptive transfer of T cells has been employed to reconstitute T cell immunity to viruses such as cytomegalovirus (CMV) in immunodeficient allogeneic stem cell transplant (SCT) patients and is being investigated to treat malignancies. In the allogeneic SCT setting, the T cells are derived from the donor and need to be isolated as clones or highly pure populations to avoid graft-versus-host disease. CD8+ T cells can be divided into defined subsets including CD62L− effector memory (TEM) and central memory T cells (TCM) expressing the CD62L lymph node homing molecule. Both TCM and TEM can give rise to cytolytic effector T cells (TE) after antigen stimulation and can be expanded in vitro for immunotherapy. However, the potential of T cells derived from either the TEM or TCM subset to persist in vivo has not been investigated. We used a macaque model to determine whether reconstitution of T cell memory to CMV by adoptive transfer of CD8+ T cell clones depended on their origin from either the CD62L+ TCM or CD62L− TEM subset. T cell clones were retrovirally transduced to express the macaque CD19 or CD20 surface marker to allow tracking of T cells in vivo. Clones derived from both TCM and TEM had similar avidity and proliferative capacity in vitro, and had a TE phenotype (CD62L−CCR7−CD28−CD127−, granzyme B+). TCM and TEM-derived T cell clones were transferred to macaques at doses of 3–6×108/kg and were both detected in the blood one day after transfer at 1.2–2.7% (low dose) to 20–25% (high dose) of CD8+ T cells. However, the frequency of TEM-derived T cells was undetectable after 3–5 days, and the cells were not present in lymph node or bone marrow obtained at day 14. By contrast, TCM-derived clones persisted in peripheral blood, migrated to tissue sites, and were detectable long-term at significant levels. A distinguishing feature of TCM-derived cells was their responsiveness to homeostatic cytokines. Only TCM-derived clones were rescued from apoptotic cell death by low-dose IL15 for >30 days in vitro and this correlated with higher levels of IL15Rα, IL2Rβ, and IL2Rγ, and of Bcl-xL and Bcl-2, which promote cell survival. To determine if the inability of TEM-derived clones to survive in vitro correlated with an increased susceptibility of cell death in vivo, we measured the proportion of infused cells that were positive for propidium iodide (PI) and Annexin V during the short period of in vivo persistence. One day after transfer, 41–45% of TEM-derived T cells were Annexin V+/PI+, analyzed directly in the blood or after 24 hours of culture. By contrast, only a minor fraction of an adoptively transferred TCM-derived T cell clone was Annexin V+/PI+ and the infused cells survived in vivo. A subset of the persisting T cells reacquired TCM marker (CD62L+CCR7+CD127+CD28+) in vivo and regained functional properties of TCM (direct lytic activity; rapid proliferation to antigen). These T cells produced IFN-γ and TNF-α after peptide stimulation, and studies are in progress to assess their in vivo response to antigen by delivery of T cells expressing CMV proteins. Our studies in a large animal model show for the first time that CD8+ TE derived from TCM but not TEM can persist long-term, occupy memory T cell niches, and restore TCM subsets of CMV-specific immunity. Thus, taking advantage of the genetic programming of cells that have become TCM might yield T cells with greater therapeutic activity and could be targeted for human studies of T cell therapy for both viral and malignant disease.

Human Alloreactive CD4+ T Cells as Potent Effector Cells and Sole Mediators of Anti-Tumor Responses in a NOD/SCID Mouse Model for Human Acute Leukemia.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1245-1245 ◽  
Author(s):  
Sanja Stevanovic ◽  
Marieke Griffioen ◽  
Marianke LJ Van Schie ◽  
Roelof Willemze ◽  
J.H. Frederik Falkenburg ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Class Ii ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
Leukemic Cells ◽  
T Cell Clones ◽  
Hla Dr ◽  
Cell Clones

Abstract Donor lymphocyte infusion (DLI) following allogeneic stem cell transplantation (alloSCT) can be a curative treatment for patients with hematological malignancies. The therapeutic benefit of DLI is attributed to a graft versus leukemia (GvL) reactivity mediated by donor T cells recognizing allo-antigens on malignant cells of the patient. Donor T cells, however, often recognize allo-antigens which are broadly expressed in non-malignant tissues of the patient, thereby causing severe graft versus host disease (GvHD). In contrast to HLA class I molecules which are ubiquitously expressed on all nucleated cells, HLA class II molecules are predominantly expressed on cells of the hematopoietic system, and therefore CD4+ T cells may selectively mediate GvL reactivity without GvHD. Several clinical studies have indeed demonstrated that CD8-depleted DLI after alloSCT can lead to clinical remissions with reduced incidence of GvHD. Since in most of these studies DLI was contaminated with CD8+ T cells, it remained unclear whether CD4+ T cells alone are capable of mediating GvL reactivity. To assess the capacity of purified CD4+ T cells to solely exert GvL reactivity we compared the anti-tumor effects of CD4+ DLI and CD3+ DLI in a NOD/SCID mouse model of human acute leukemia. Iv injection of primary human leukemic cells from three different patients reproducibly resulted in engraftment of leukemia in mice, as monitored by peripheral blood analysis. Three weeks after inoculation of leukemic cells, established tumors were treated by infusion of human donor T cells. In mice treated with CD4+ DLI (5*106 CD4+ T cells), the emergence of activated (HLA-DR+) T cells coincided with rapid disappearance of leukemic cells, showing similar kinetics as for CD3+ DLI (consisting of 5*106 CD4+ T cells and 3*106 CD8+ T cells). To analyze the specific reactivity of T cells responsible for the anti-leukemic effect, we clonally isolated human CD45+ T cells during the anti-tumor response following CD4+ DLI in which the donor was matched for HLA class I and mismatched for the HLA-DR (DRB1*1301), -DQ (DQB1*0603) and –DP (DPB1*0301/0401) alleles of the patient. A total number of 134 CD4+ T cell clones were isolated expressing various different TCR Vbeta chains. Most of the isolated CD4+ T cell clones (84%) were shown to be alloreactive, as determined by differential recognition of patient and donor EBV-transformed B cells (EBV-LCL) in IFN-g ELISA. A substantial number of these CD4+ T cell clones also exerted cytolytic activity (17%), as demonstrated by specific reactivity with patient EBV-LCL but not donor EBV-LCL in a 10 hr 51Cr-release cytotoxicity assay. Further characterization of the specificity of 20 CD4+ T cell clones using blocking studies with HLA class II specific monoclonal antibodies illustrated HLA class II restricted recognition directed against HLA-DR (n=3), HLA-DQ (n=16) and HLA-DP (n=1) molecules of the patient. Of the 127 alloreactive CD4+ T cell clones, only 36 clones directly recognized primary leukemic cells of the patient. Flowcytometric analysis demonstrated that HLA class II, and in particular HLA-DQ, molecules were expressed at relatively low levels on patient leukemic cells as compared to patient EBV-LCL. Upregulation of HLA class II and costimulatory molecules on patient leukemic cells upon differentiation in vitro into leukemic antigen presenting cells (APC) resulted in recognition of patient leukemic cells by all alloreactive CD4+ T cell clones. Therefore, we hypothesize that the alloreactive CD4+ T cells have been induced in vivo by patient leukemic cells, which, upon interaction with T cells or other environmental factors, acquired an APC phenotype. In conclusion, our data show that alloreactive CD4+ T cells can be potent effector cells and sole mediators of strong antitumor responses in a NOD/SCID mouse model for human acute leukemia.

Identification and Characterization of Human Aspergillus Fumigatus-Specific Tr1-(Like) Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 181-181
Author(s):  
Tanja Bedke ◽  
Sarah Lurati ◽  
Claudia Stuehler ◽  
Nina Khanna ◽  
Hermann Einsele ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Regulatory T Cells ◽  
Aspergillus Fumigatus ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Ifn Gamma ◽  
T Cell Clones ◽  
Suppressor T Cell ◽  
Cell Clones

Abstract Abstract 181 Introduction: The ubiquitous mold Aspergillus fumigatus (A. fumigatus) induces two forms of pathogenesis: invasive aspergillosis in neutropenic patients and allergic aspergillosis in patients with chronic obstructive lung disease as well as in immunosuppressed patients. Mouse models of aspergillosis suggest that not only effector T cells (Teff) but also regulatory T cells (Treg) play a crucial role for the regulation of a protective T cell-mediated immunity to A. fumigatus. However, it is little-known about the involvement of Treg during A. fumigatus infection in humans. In order to develop new therapeutical strategies for the treatment of aspergillosis this project aims to understand the influence of regulatory T cells on A. fumigatus infection in humans. Material/Methods: A. fumigatus-specific CD4+ T cell clones were established from PBMC of healthy donors. Based on this clone pool Treg clones were identified due to their inability to proliferate in the absence of costimulation assessed by 3[H]-TdR incorporation as well as their Ag-specific cytokine production and phenotype determined by flow cytometry. Treg function was analyzed by their ability to suppress proliferation of autologous CD4+ T cells using CFSE dilution. Results: We identified A. fumigatus-specific T cell clones that exhibited marginal detectable proliferation after restimulation with immobilized anti-CD3 mAb in the absence of costimulation. However, these T cell clones vigorously proliferated in response to restimulation with their cognate antigen. A more detailed characterization showed that these suppressor T cell clones produced high amounts of IL-10 and moderate levels of IFN-gamma upon Ag-specific restimulation and expressed low amounts of Foxp3 but not Helios, a transcription factor that had recently been linked to natural occurring Treg. Most importantly, these T cell clones suppressed Ag-specific expansion of CD4+ Teff. This effect was contact-independent since suppression of Ag-specific CD4+ T cell expansion detected in transwell experiments was comparable to cocultures that enabled cellular-contact. Furthermore, anti-CD3/CD28-induced proliferation of naïve CD4+ T cells was not reduced in the presence of culture supernatants obtained from suppressor T cell clones after their antigen-specific restimulation in the absence of DCs. Conclusions: We identified for the first time A. fumigatus-specific CD4+ T cell clones with a Tr1(-like) IL-10+IFN-gamma+Foxp3lowHelios− phenotype. These cells suppressed expansion of A. fumigatus-specific Teff in an Ag-specific manner mediated by soluble factors released from Tr1(-like) cell clones. Since these factors did not affect CD4+ T cell proliferation in the absence of DCs our data suggest, that Tr1(-like) cell clones rather negatively regulate the stimulatory capacity of DCs leading to a reduced expansion of Ag-specific CD4+ T cells. Therefore these Tr1(-like) cells might play a protective role during A. fumigatus infection in humans. Thus, adoptive transfer of A. fumigatus-specific Treg could be useful to enhance protective immunity in patients with chronic A. fumigatus infection. Disclosures: Topp: Micromet: Consultancy, Honoraria.

Graft Versus Leukemia Separates From Graft Versus Host Disease By Magnitude and Avidity Of The Allo-Reactive T Cell Response After Allogeneic Stem Cell Transplantation and Donor Lymphocyte Infusion

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2014-2014
Author(s):  
Cornelis A.M. van Bergen ◽  
Simone A.P. Van Luxemburg-Heijs ◽  
Matthijs Eefting ◽  
Maria W. Honders ◽  
Inge Jedema ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cell Response ◽  
T Cell Response ◽  
Functional Avidity ◽  
T Cell Clones ◽  
Inflammatory Conditions ◽  
Hla Dr ◽  
Cell Clones

Abstract Donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation (alloSCT) can be a curative treatment for patients with hematological malignancies due to the capacity of allo-reactive donor derived T cells to mediate a curative potent graft versus leukemia (GVL) effect. However, associated acute graft versus host disease (GVHD) remains a major risk. To study the role of CD8+ T cells in GVL reactivity and GVHD, we selected patients who responded to DLI (without preceding cytoreductive treatment) for recurrent disease or incomplete donor chimerism after alloSCT. The patients were grouped according to absence (7 patients) or presence (6 patients) of GVHD. To quantify the number of circulating activated CD8+ T cells before DLI and at the time of disease regression or conversion to full donor chimerism we measured the frequencies of CD8+ HLA-DR+ T cells in peripheral blood samples by flowcytometry. Before DLI, highly variable numbers of CD8+ HLA-DR+ T cells were found (37.8 ± 42.9 x106/L), that significantly increased after DLI (309±473 x106/L, p<0.005), demonstrating involvement of CD8+ HLA-DR+ T cells in immune responses after DLI. To determine the specificity and functional avidity of the CD8+ HLA-DR+ T cells, these cells were isolated using flowcytometric cell sorting and clonally expanded. From a total of 30 samples, on average 225 T cell clones per sample were obtained and tested for recognition of patient and donor derived EBV-LCL, CD40L stimulated B cells (CD40L-B cells) and monocyte derived dendritic cells (monoDC). Surprisingly, in many samples from both patient cohorts high percentages of clones recognizing EBV-LCL derived from both patient and donor but not recognizing CD40L-B cells and monoDC were found. These T cells may be involved in anti-EBV responses irrespective of the presence of a GVL effect or GVHD. To investigate whether the magnitude of the allo-immune response was different in patients with or without GVHD coinciding the GVL effect, we compared the frequencies of allo-reactive T cell clones in samples from both patient groups. Significantly lower percentages of allo-reactive T cell clones were found in patients without GVHD as compared to patients with GVHD (5.1 ± 7.0% versus 32.5 ± 20.0% respectively, p<0.01), showing that coinciding GVHD is associated with an increased magnitude of the allo-reactive T cell response. Per patient, we determined the number of unique antigens targeted by the isolated T cell clones by characterizing the targeted MiHA using whole genome association scanning. In line with the lower total number of allo-reactive T cells, a lower number of unique MiHA was targeted in patients without GVHD (2.7±3.5) as compared to patients with GVHD (10.2±5.8, p=0.015). To determine whether occurrence of GVHD could be explained by the tissue specificity and functional avidity of the allo-reactive T cell response after DLI, we tested the T cell clones obtained from both patient cohorts for recognition of fibroblasts (FB) derived from skin biopsies of the patient. To mimic pro-inflammatory conditions, FB were pretreated for 4 days with 100 IU/ml IFN-γ. Recognition of untreated FB was exclusively mediated by T cell clones obtained from patients with GVHD, whereas recognition of IFN-γ pretreated FB was found for clones isolated from patients with or without coinciding GVHD. In addition, several T cell clones isolated from patients without GVHD were found to be directed against MiHA encoded by genes with a broad expression profile in non-hematopoietic cells comprising FB, despite absence of FB recognition under non-inflammatory conditions. This suggests that in addition to the tissue expression profile of the MiHA other factors, comprising the local inflammatory milieu, play a role in the risk of developing GVHD. In conclusion, our data show a strong correlation between the magnitude and the functional avidity of the allo-reactive CD8+ T cell response and the occurrence of GVHD after DLI. We hypothesize that the limited production of pro-inflammatory cytokines due to the moderate magnitude of the immune response in patients mounting a GVL response without coinciding GVHD reactivity may have prevented the induction of GVHD by the lower avidity allo-reactive T cells, that under pro inflammatory conditions can mediate GVHD by recognition of normal non-hematopoietic cells of the patient. Disclosures: No relevant conflicts of interest to declare.

CD4+ T-cell clones recognizing human lymphoma-associated antigens: generation by in vitro stimulation with autologous Epstein-Barr virus–transformed B cells

Blood ◽  
2009 ◽  
Vol 114 (4) ◽  
pp. 807-815 ◽  
Author(s):  
Heather M. Long ◽  
Jianmin Zuo ◽  
Alison M. Leese ◽  
Nancy H. Gudgeon ◽  
Hui Jia ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Epstein Barr Virus ◽  
Hla Class Ii ◽  
Cd4 T Cell ◽  
T Cell Clones ◽  
Barr Virus ◽  
Cell Clones ◽  
Epstein Barr

Abstract Epstein-Barr virus (EBV)–specific T-cell preparations, generated by stimulating immune donor lymphocytes with the autologous virus-transformed B-lymphoblastoid cell line (LCL) in vitro, can be used to target EBV-positive malignancies. Although these preparations are enriched for EBV antigen–specific CD8+ T cells, most also contain a CD4+ T-cell population whose specificity is unknown. Here, we show that, although CD4+ T-cell clones derived from such cultures recognize HLA class II–matched LCLs but not mitogen-activated B lymphoblasts, many (1) do not map to any known EBV antigen, (2) can be raised from EBV-naive as well as EBV-immune persons, and (3) can recognize a broad range of human B lymphoma–derived cell lines irrespective of EBV genome status, providing those lines to express the relevant HLA class II–restricting allele. Importantly, such CD4+ clones not only produce IFNγ but are also cytotoxic and can control the outgrowth of HLA-matched lymphoma cells in cocultivation assays. We infer that such CD4+ T cells recognize cellular antigens that are preferentially up-regulated in EBV-transformed but not mitogen-activated B lymphoblasts and that are also expressed in a range of B-cell malignancies. Such antigens are therefore of potential value as targets for CD4+ T cell–based immunotherapy.

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