Immature dendritic cells (imDCs) can have a tolerizing effect under normal conditions or after transplantation. However, because of the significant heterogeneity of this cell population, it is extremely difficult to study the mechanisms that mediate the tolerance induced or to harness the application of imDCs for clinical use. In the present study, we describe the generation of a highly defined population of imDCs from hematopoietic progenitors and the direct visualization of the fate of TCR-transgenic alloreactive CD4 ؉ and CD8 ؉ T cells after encountering cognate or noncognate imDCs. Whereas CD4 ؉ T cells were deleted via an MHC-independent mechanism through the NO system, CD8 ؉ T-cell deletion was found to occur through a unique MHCdependent, perforin-based killing mechanism involving activation of TLR7 and signaling through Triggering Receptor-1 Expressed on Myeloid cells (TREM-1). This novel subpopulation of perforinexpressing imDCs was also detected in various lymphoid tissues in normal animals and its frequency was markedly enhanced after GM-CSF administration. IntroductionCentral and peripheral tolerance mechanisms are critical for the establishment of a robust immune response that can distinguish between self-and nonself-antigens. Although the majority of self-specific T cells are deleted by negative selection in the thymus, some self-reactive T cells are spared and can reach peripheral organs. 1,2 A wealth of evidence indicates that dendritic cells (DCs) have tolerogenic capacity in their immature state (imDCs). [3][4][5][6][7][8] In the context of allogeneic organ transplantation, infusion of imDCs expressing the relevant MHC-peptide complex can prolong allograft survival in vivo. 7,[9][10][11][12] At the same time, imDCs can become immunogenic on maturation/activation in the presence of a danger signal such as lipopolysaccharide (LPS). However, this simplistic paradigm was recently challenged by the demonstration that fully mature DCs can also induce tolerance under the appropriate conditions, [13][14][15][16][17] suggesting a more complex decision-making process in which the net effect of Ag dose, DC lineage, DC maturation and activation state, and the cytokine milieu at the site of inflammation determine whether immunogenic or tolerogenic DC activity will prevail. 18 The tolerogenic potential of immature or mature DCs can be further extended to the resolution of inflammatory responses to pathogens. 19 Lymphoid organs, including spleen, bone marrow (BM), lymph nodes, and thymus, contain multiple DC subpopulations largely defined by their distinct anatomical location and phenotypes. 20,21 For example, the mouse spleen harbors plasmacytoid DCs (pDCs) and the CD8 ϩ and CD8 Ϫ subsets of classic DCs (cDCs). 20 Apart from the major steady-state dichotomy of differentiation into pDCs versus cDCs, an additional distinct monocyte-derived DC subset with phenotypic characteristics of cDCs is recruited to sites of inflammation. 22 The phenotypic heterogeneity of DCs and growing data on their distinct origins p...
Cytotoxic T lymphocytes (CTLs) suppress T cell responses directed against their antigens regardless of their own T cell receptor (TCR) specificity. This makes the use of CTLs promising for tolerance induction in autoimmunity and transplantation. It has been established that binding of the CTL CD8 molecule to the major histocompatibility complex (MHC) class I ␣3 domain of the recognizing T cell must be permitted for death of the latter cell to ensue. However, the signaling events triggered in the CTL by this molecular interaction in the absence of TCR recognition have never been clarified. Here we use single-cell imaging to study the events occurring in CTLs serving as targets for recognition by specific T cells. We demonstrate that CTLs actively respond to recognition by polarizing their cytotoxic granules to the contact area, releasing their lethal cargo, and vigorously proliferating. Using CTLs from perforin knockout (KO) mice and lymphocyte specific kinase (Lck) knockdown with specific small interfering RNA (siRNA), we show that the killing of the recognizing CD8 T cell is perforin dependent and is initiated by Lck signaling in the CTL. Collectively, these data suggest a novel mechanism in which the entire cascade generally triggered by TCR engagement is "hijacked" in CTLs serving as targets for T cell recognition without TCR ligation. (Blood. 2011;117(3):1042-1052) IntroductionCTLs recognize and kill target cells with marked specificity. This specificity is conferred on them by their TCR, which recognizes peptides in the context of target cell major histocompatibility complex class I (MHC-I). 1-3 However, CTLs can induce death in a manner that does not involve their TCR. This occurs when CTLs suppress immune responses directed against their antigens in an activity coined "veto." 4 This type of CTL activity is also of a specific nature, as only T cells carrying TCRs capable of recognizing MHC-peptide (MHC-p) complexes displayed by the CTL are killed. 4,5 This unique type of CTL activity has been heavily studied in the context of transplantation [5][6][7][8][9][10] because CTLs can eliminate alloreactive T cells directed against them, and consequently against tissues carrying identical MHC-p complexes, without harming beneficial T cells directed against pathogens, thus inducing specific tolerance toward transplanted tissue. 6,11 However, veto activity in CTLs is not necessarily limited to the allogeneic setting. Indeed, suppression of specific antipeptide responses by peptide-presenting CTLs has been demonstrated in the syngeneic setting. 12 Thus, it has been suggested that CTLs may be important for maintaining selftolerance by suppressing autoreactive T cell responses. 8,12 In studies assaying this inhibitory CTL activity, it has been demonstrated that for killing to occur, the recognizing T cell must be allowed to contact the CTL. 5,9,[13][14][15][16] Uniquely, the CD8 molecule of the CTL must be allowed to engage nonpolymorphic residues of the ␣3 domain of the recognizing T cell MHC-I molecule. 5,12,17 Howe...
Key Points• A new approach to achieving immune tolerance and mixed chimerism with relevance for hematopoietic stem cell and organ transplantation. IntroductionHematopoietic stem cell transplantation (HSCT) can offer a cure for patients with a variety of nonmalignant hematologic disorders, such as sickle cell anemia and thalassemia. Furthermore, considering that chimerism induction is generally associated with immune tolerance to donor antigens 1-4 it can serve as a platform for immune tolerance induction to be followed by either solid organ transplantation or by continuous cell therapy with donor cells in cancer patients. Therefore, attaining a well-tolerated HSCT protocol represents one of the most desirable goals in transplantation biology. However, the high risk for infections and GVHD, linked with procedures currently used in HSCT in leukemia patients, are not acceptable for patients with nonmalignant diseases associated with longer life expectancy. Thus, developing new, safer approaches for achieving hematopoietic chimerism under milder conditioning than that used in leukemia patients, and with reduced risk for GVHD, is of utmost importance.The problem of GVHD could be adequately addressed, even in mismatched haploidentical transplants, by rigorous T-cell depletion or positive selection of CD34 ϩ hematopoietic stem cells (HSCs), using megadoses of HSCs to overcome the residual host immunity remaining after myeloablative conditioning. This immune modulation is mediated, at least partially, by virtue of potent veto activity exhibited by CD34 ϩ HSCs and their early myeloid derivatives [5][6][7] which rapidly expand during the early posttransplant period. Veto activity, was defined as the capacity to specifically suppress CTL-precursor (CTLp) cells, directed against antigens (Ags) expressed by the veto cells. Therefore, use of donor-derived veto cells as specific immunosuppressants in transplantation settings, There is an Inside Blood commentary on this article in this issue.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on June 7, 2019. by guest www.bloodjournal.org From eliminating only host anti-donor T cells while sparing others, is highly attractive. However, the number of CD34 ϩ HSC that can be harvested is limited, and insufficient for overcoming the robust host immunity surviving reduced-intensity conditioning (RIC; Gan et al, unpublished results). Therefore, combining megadoses of CD34 ϩ HSC transplantation (HSCT) with other tolerizing veto cells could potentially support and promote successful engraftment of purified HSCs under a safer RIC protocol. One approach to address this challenge could be provided using donor CD8 T cells, shown to be endowed with the most potent veto activity. 8 However, the utility of these cells for tolerance induction is l...
We previously demonstrated that anti–third-party CTLs (stimulated under IL-2 deprivation against cells with an MHC class I [MHC-I] background different from that of the host and the donor) are depleted of graft-versus-host reactivity and can eradicate B cell chronic lymphocytic leukemia cells in vitro or in an HU/SCID mouse model. We demonstrated in the current study that human allogeneic or autologous anti–third-party CTLs can also efficiently eradicate primary non-Hodgkin B cell lymphoma by inducing slow apoptosis of the pathological cells. Using MHC-I mutant cell line as target cells, which are unrecognizable by the CTL TCR, we demonstrated directly that this killing is TCR independent. Strikingly, this unique TCR-independent killing is induced through lymphoma MHC-I engagement. We further showed that this killing mechanism begins with durable conjugate formation between the CTLs and the tumor cells, through rapid binding of tumor ICAM-1 to the CTL LFA-1 molecule. This conjugation is followed by a slower second step of MHC-I–dependent apoptosis, requiring the binding of the MHC-I α2/3 C region on tumor cells to the CTL CD8 molecule for killing to ensue. By comparing CTL-mediated killing of Daudi lymphoma cells (lacking surface MHC-I expression) to Daudi cells with reconstituted surface MHC-I, we demonstrated directly for the first time to our knowledge, in vitro and in vivo, a novel role for MHC-I in the induction of lymphoma cell apoptosis by CTLs. Additionally, by using different knockout and transgenic strains, we further showed that mouse anti–third-party CTLs also kill lymphoma cells using similar unique TCR-independence mechanism as human CTLs, while sparing normal naive B cells.
Transplantation of T cell-depleted BM (TDBM) under mild conditioning, associated with minimal toxicity and reduced risk of GVHD, offers an attractive therapeutic option for patients with nonmalignant hematologic disorders or can be used to induce immune tolerance to subsequent organ transplantation. However, overcoming TDBM rejection after reduced conditioning remains a challenge. We previously demonstrated that this barrier can be overcome using donor-derived naïve CD8 T cells if they are expanded against third-party alloantigens under culture conditions inducing a central memory phenotype. Thus, such anti-3rd party CD8 T cells (Tcm) exhibit marked veto activity and reduced risk for GVHD due to the lower frequency of anti-host clones achieved during the culture period. In the present study, we tested the feasibility of generating veto Tcm by stimulation against well-defined peptides including viral antigens. For proof of concept, we used OT1 mice that express a transgenic (Tg) TCR designed to recognize ovalbumin (OVA) residues 257-264 in the context of H2Kb MHC-I. Prior to harvest of OT1 CD8+ T cells, mice were immunized twice with OVA-peptide. Mice were sacrificed 7 to 14 days after immunization, their spleens and lymph nodes removed and crushed, and magnetic bead sorting utilized to isolate the memory cells (CD8+CD44+). The resulting population was subjected to third-party stimulation by co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice, under cytokine deprivation. hIL-15 (10ng/ml) was added to the culture 60 hrs after culture initiation to induce the cells to express a Tcm like phenotype, as previously described for anti-3rd party Tcm generated from WT naïve CD8 T cells (WT Tcm). When tested in vivo, the OT-1 Tcm (H2Kb) were able to enhance engraftment of allogeneic T cell depleted BMT (H2Kb) in sub-lethally irradiated (5.5 Gy TBI) Balb/c recipients (H2Kd), in analogy to the chimerism induced by WT Tcm. This initial successful experiment was then followed by an experiment, which more closely resembled the human setting, in which the CD8+CD44+ cells were isolated from WT C57BL/6 OVA-immunized mice and subsequently introduced to co-culture with irradiated splenocytes generated from spleens of OVA-expressing mice. Results showed that Tcm grown from such a starting population of CD8+CD44+ cells were also able to achieve marked donor chimerism when administered with megadose of T cell depleted (TCD) alloSCT in sublethally irradiated Balb/c recipients (Fig.1A). Notably, infusion of 5x106 Tcm into sublethally irradiated mice (5.5. GY TBI) without BMT, did not cause any GVHD as measured by weight loss, in contrast to CD8+CD44+ T cells used to generate the Tcm (Fig.1B). These results demonstrate that CD8+CD44+ derived from memory CD8 T cells by expansion against cognate peptides exhibit markedly reduced risk for GVHD compared to freshly isolated memory cells, while retaining their veto activity and inducing tolerance. Finally, based on these proof of concept studies, we were able to translate this approach and generate human anti-viral CD8 veto cells with central memory phenotype. Thus, CD8+CD45RO+ memory T cells were selected by depletion of CD4+CD56+CD45RA+ cells from PBMC of normal donors and then co-cultured with donor DCs pulsed with a viral peptide mixture of three prominent viruses (EBV, CMV and Adenovirus). In three large scale experiments using leukapheresis preparations of normal CMV and EBV positive donors, with GMP grade reagents, more than 1x109 Tcm could be attained by the end of 9 days of culture (average expansion of Tcm=13.5±4 fold) with greater than 90% purity of CD45RO+CD62L+CD8+ T cells (Fig 2). The harvested anti-viral Tcm exhibited more than 3 log depletion of alloreactivity, compared to fresh CD8 T cells, as measured by limit dilution analysis of cytotoxic T cell precursors against host type target cells (Fig. 3). In conclusion, our results suggest that potent veto CD8 Tcm can be generated from the memory pool of donors positive for viral reactivity by stimulation against viral antigens. Such veto Tcm could be most attractive for haploidenitcal transplantation of TCDBM, as they can enable engraftment following non-myeloablative conditioning and at the same time provide anti-viral protection. *E.B.L and N.O.G contributed equally Disclosures Bachar Lustig: Yeda LTD: Patents & Royalties. Or Geva:Yeda LTD: Patents & Royalties. Lask:Yeda LTD: Patents & Royalties. Gidron:Yeda LTD: Patents & Royalties. Kagan:Yeda LTD: Patents & Royalties. Reisner:Cell Source LTD: Consultancy, Equity Ownership, Patents & Royalties, Research Funding.
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