Donor-derived Antigen Presenting Cells Research Articles

The potential role of infusions of T regulatory cells in inducing and maintaining liver allograft tolerance.

1_nse8422sKaltura.

IFN-γR/STAT1 signaling in recipient hematopoietic antigen-presenting cells suppresses graft-versus-host disease.

The absence of IFN-γ receptor (IFN-γR) or STAT1 signaling in donor cells has been shown to result in reduced induction of acute graft-versus-host disease (GVHD). In this study, we unexpectedly observed increased activation and expansion of donor lymphocytes in both lymphohematopoietic organs and GVHD target tissues of IFN-γR/STAT1-deficient recipient mice, leading to rapid mortality following the induction of GVHD. LPS-matured, BM-derived Ifngr1-/- Stat1-/- DCs (BMDCs) were more potent allogeneic stimulators and expressed increased levels of MHC II and costimulatory molecules. Similar effects were observed in human antigen-presenting cells (APCs) with knockdown of Stat1 by CRISPR/Cas9 and treatment with a JAK1/2 inhibitor. Furthermore, we demonstrated that the absence of IFN-γR/STAT1 signaling in hematopoietic APCs impaired the presentation of exogenous antigens, while promoting the presentation of endogenous antigens. Thus, the indirect presentation of host antigens to donor lymphocytes was defective in IFN-γR/STAT1-deficient, donor-derived APCs in fully donor chimeric mice. The differential effects of IFN-γR/STAT1 signaling on endogenous and exogenous antigen presentation could provide further insight into the roles of the IFN-γ/STAT1 signaling pathway in the pathogenesis of GVHD, organ rejection, and autoimmune diseases.

Natural Killer Cells are Essential for Allograft Vasculopathy in a Model of CD4+ T Cell-Mediated Chronic Rejection

Purpose In transplantation, recipient CD4+ T cells have two potential means of MHC class II-restricted donor antigen detection: (1) the direct recognition of allogeneic MHC class II expressed by donor antigen-presenting cells (APCs) and (2) the indirect recognition of donor antigens processed and presented by recipient APCs. As donor-derived APCs are gradually depleted over time, the indirect pathway emerges as the dominant mode of allorecognition in the long-term. A clear role of direct CD4+ T cells as effector cells has been shown in acute rejection, however, the role of indirect CD4+ T cells in chronic rejection is less clear. The function of NK cells in solid organ transplantation is multifaceted, but they have been implicated in several different animal models of chronic rejection. Our study sought to test the hypothesis that NK cells are the key effectors in sustaining an anti-donor response in a model of indirect CD4+ T cell mediated chronic allograft vasculopathy (CAV). Methods We utilized a mouse model of cardiac transplantation with MHC class II-deficient B6.C2D (H-2b) hearts engrafted into immune-deficient BALB/c.rag-/- (H-2d) recipients. Recipients had no functional adaptive immunity but an intact innate immune system. After transplantation, the recipients received purified polyclonal BALB/c CD4+ T cells with or without concurrent administration of an NK cell depleting antibody (anti-Asialo GM1). Allografts and recipient spleens were subsequently assessed after 30 days. Results All allografts demonstrated cardiac contraction, as detected by abdominal palpation, for 30 days both with and without CD4+ T cell reconstitution. Only 1/9 (11%) of allografts from un-reconstituted recipients demonstrated evidence of CAV, while 5/7 (72%) of allografts from recipients that received adoptively transferred CD4+ T cells showed disease (p Conclusion In the context of immunodeficient hosts, CD4+ T cells are sufficient to mediate chronic allograft vasculopathy, independent of recipient CD8+ T cell and B cell responses. Recipient NK cells are necessary for the development of this form of chronic rejection.

Neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance.

Neutrophil extracellular traps (NETs) have been shown to worsen acute pulmonary injury including after lung transplantation. The breakdown of NETs by DNAse-1 can help restore lung function, but whether there is an impact on allograft tolerance remains less clear. Using intravital 2-photon microscopy, we analyzed the effects of DNAse-1 on NETs in mouse orthotopic lung allografts damaged by ischemia-reperfusion injury. Although DNAse-1 treatment rapidly degrades intragraft NETs, the consequential release of NET fragments induces prolonged interactions between infiltrating CD4+ T cells and donor-derived antigen presenting cells. DNAse-1 generated NET fragments also promote human alveolar macrophage inflammatory cytokine production and prime dendritic cells for alloantigen-specific CD4+ T cell proliferation through activating toll-like receptor (TLR) - Myeloid Differentiation Primary Response 88 (MyD88) signaling pathways. Furthermore, and in contrast to allograft recipients with a deficiency in NET generation due to a neutrophil-specific ablation of Protein Arginine Deiminase 4 (PAD4), DNAse-1 administration to wild-type recipients promotes the recognition of allo- and self-antigens and prevents immunosuppression-mediated lung allograft acceptance through a MyD88-dependent pathway. Taken together, these data show that the rapid catalytic release of NET fragments promotes innate immune responses that prevent lung transplant tolerance.

OR2 Recipient NK cell receptor repertoire comprising activating KIR2DS5 and licensed KIR3DL1 is associated with improved chronic lung allograft dysfunction (CLAD) free survival when donor misses HLA-BW4 ligand

Aim Alloimmune responses manifesting as chronic lung allograft dysfunction (CLAD) limit long-term survival of lung transplant. Natural killer (NK) cells expressing an inhibitory KIR (iKIR) recognizing self-HLA can be activated when confronted with allograft lacking a ligand for that iKIR. Here we investigated if allograft missing ligand for recipient iKIR that had a cognate HLA ligand would enhance CLAD-free survival. We also analyzed the role of recipient’s activating KIRs (aKIR) on CLAD-free survival. Methods Using luminex rSSO methods, KIR and HLA genes were typed for 263 lung transplant recipients and HLA genes were typed for all donors. CLAD was determined by date of FEV1 or FVC drop to KIR-HLA genotypes. Results Bx KIR genotype recipients (i.e., carrying more a KIR s) had higher median CLAD-free survival than those carrying AA genotypes (Fig. A). Recipients with all 5 B haplotype-associated a KIR s ( 2DS1, S2, S3, S5, 3DS1 ) had enhanced CLAD-free survival relative to those missing one or more of these a KIR s. Particularly, recipients carrying 2DS5 showed enhanced CLAD-free survivals than those missing 2DS5 (Fig. B). Further, CLAD-free survival was higher for recipients carrying KIR3DL1 + Bw4 when donors missing Bw4 ligand (Fig. C). Allografts missing relevant ligands for recipients carrying 2DL1 + C2, 2DL2/L3 + C1, and/or 3DL2 + A3/A11 pairs were not associated with differential survival. Recipients with 2DS5, 3DL1, and Bw4 receiving donors missing Bw4 ligand showed the longest CLAD-free survival (p = 0.01, 9.2 median survival years; Fig. D). Conclusions Recipient NK cells expressing aKIRs and/or licensed KIR3DL1 may respond and deplete donor-derived antigen presenting cells that do not express Bw4 ligand, and thus impair direct allorecognition pathways which in turn delay CLAD. Functional studies will be required to understand the mechanism of KIR-HLA interactions in protecting from CLAD.

T Cells Motility in the Colonic Gvhd Is Influenced By Both Cognate Interaction and Microenvironment

Graft-versus-host disease (GVHD) accounts for substantial morbidity and mortality after allogeneic bone marrow transplantation. Allograft T cells are stimulated by both host and donor-derived antigen presenting cells (APCs). After differentiating into T effector cells, donor T cells migrate to GVHD target organs where they mediate damage directly and indirectly. However, very little is known about the dynamics of T cells in GVHD target tissues and how their behaviors are affected by the local environments, including by tissue-resident APCs. We utilized two-photon intravital microscopy (2PIM) to analyze T cell locations, motility and interactions with APCs. 129S1/SVLmJ (129) (H-2b) hosts were irradiated and reconstituted with C57Bl/6 (B6) (H-2b) CD11-YFP transgenic mice bone marrow together with B6 dsRed (RFP) CD8 cells and cyan fluorescent protein (CFP) CD4 cells. Mice were imaged from days 25-42 post-transplant. We focused on the colon which is strongly targeted in this model and in humans and which previously has been difficult to image by 2PIM. In syngeneic B6 into B6 recipients, donor CD11c+, CD8+ and CD4+ cells populate the colonic mucosa. However, a greater number of cells accumulated in the allogenic as compared to syngeneic recipients. Based on the analysis of 7-8 mice per group, T cells in allogenic mice were less motile. The mean CD8 speeds in syngeneic and allogeneic mice were 3.38 ± 0.07 µm/min and 2.21 ± 0.03 µm/min, respectively. Mean CD4 speeds in syngeneic and allogeneic mice were 3.11 ± 0.08 µm/min and 2.38 ± 0.04 µm/min, respectively. The majority of T cells were stationary, with few entering or leaving the imaged volume. Perfusion was confirmed by i.v. rhodamine-dextran; therefore, stationary behaviors was not due to tissue viability. Using optical clearing we imaged whole-mount colonic tissue from mucosa to serosa. T cells were aggregated in the sub-mucosa and infiltrated crypts and surprisingly also the muscular layer. Ki67+ T cells were found throughout, especially in the submucosa. Given the 2PIM data which show few new T cells entering the imaged volumes, these cells were likely stimulated to divide locally. Preliminary data with EdU pulsing suggest this to be the case. To determine whether T cell stability is microenvironment- or antigen-driven, we injected OT-1 TCR transgenic effectors which do not recognize any antigen in the recipient and imaged the next day. Most colonic OT-1 cells showed the same stationary behavior as nearby donor-derived CD4 and CD8 cells, suggesting that factors in the GVHD colon microenvironment drive T cell stability. Nonetheless there was also an antigen-driven component as injection of an anti-MHCII antibody (but not isotype control Ab) increased CD4+ T cell motility presumably by disrupting TCR:MHCII interaction. These results show the motility of T cells in the GVHD colon is influence by both TCR:MHC cognate interactions and by the microenvironment. That T cells are dividing and may be activated in situ, suggesting GVHD may be maintained locally. Current studies are focusing on what antigen-independent factors affect T cell motility and on defining the roles played by tissue APCs. DisclosuresNo relevant conflicts of interest to declare.

Role of C-C motif ligand 2 and C-C motif receptor 2 in murine pulmonary graft-versus-host disease after lipopolysaccharide inhalations.

Environmental exposures are a potential trigger of chronic pulmonary graft-versus-host disease (pGVHD) after successful recovery from hematopoietic cell transplant (HCT). We hypothesized that inhalations of LPS, a prototypic environmental stimulus, trigger pGVHD via increased pulmonary recruitment of donor-derived antigen-presenting cells (APCs) through the C-C motif ligand 2 (CCL2)-C-C motif receptor 2 (CCR2) chemokine axis. B10.BR(H2(k)) and C57BL/6(H2(b)) mice underwent allogeneic (Allo) or syngeneic (Syn) HCT with wild-type (WT) C57BL/6, CCL2(-/-), or CCR2(-/-) donors. After 4 weeks, recipient mice received daily inhaled LPS for 5 days and were killed at multiple time points. Allo mice exposed to repeated inhaled LPS developed prominent lymphocytic bronchiolitis, similar to human pGVHD. The increase in pulmonary T cells in Allo mice after LPS exposures was accompanied by increased CCL2, CCR2, and Type-1 T-helper cytokines as well as by monocytes and monocyte-derived dendritic cells (moDCs) compared with Syn and nontransplanted controls. Using CCL2(-/-) donors leads to a significant decrease in lung DCs but to only mildly reduced CD4 T cells. Using CCR2(-/-) donors significantly reduces lung DCs and moDCs but does not change T cells. CCL2 or CCR2 deficiency does not alter pGVHD pathology but increases airway hyperreactivity and IL-5 or IL-13 cytokines. Our results show that hematopoietic donor-derived CCL2 and CCR2 regulate recruitment of APCs to the Allo lung after LPS exposure. Although they do not alter pathologic pGVHD, their absence is associated with increased airway hyperreactivity and IL-5 and IL-13 cytokines. These results suggest that the APC changes that result from CCL2-CCR2 blockade may have unexpected effects on T cell differentiation and physiologic outcomes in HCT.

Specific increase in T cell potency via structure-based design of a T cell receptor for adoptive immunotherapy

Adoptive immunotherapy with antigen-specific T lymphocytes is a powerful strategy for cancer treatment. However, most tumor antigens are non-reactive self proteins, which presents an immunotherapy design challenge. Studies have shown that tumor-specific T cell receptors (TCRs) can be transduced into normal peripheral blood lymphocytes, which persist after transfer in about 30% of patients and effectively destroy tumor cells. Still, recent clinical trial with affinity-enhanced TCRs has resulted in severe effects due to cross reactivity to an unrelated peptide. Thus, the challenge for targeted T cell therapy remains to increase T cell potency in order to improve clinical responses and ensure on-target specificity by avoiding unwanted cross reactivity. We used structure-based design to predict point mutations of a TCR (DMF5) that enhance its binding affinity for an agonist tumor differentiation antigen-major histocompatibility complex (pMHC), Mart-1(27L)-HLA-A2, which elicits full T cell activation to trigger immune responses. Structural based approaches have been used to increase TCR affinity, however their potential cross-reactivity has not been reported. Here, we analyzed the effects of selected TCR point mutations alone and in combination on T cell activation potency. Further, we analyzed their specificity and cross-reactivity with related antigens presented by different melanoma cell lines and donor-derived antigen presenting cells. Our structure-based approach allowed us to rationally design sequence substitutions that improve binding in contact areas between the TCR and pMHC without increasing cross-reactivity with a wide variety of self-antigens. We identified and evaluated point mutations in critical TCR positions resulting in more potent T cell activation but maintaining overall specificity. When double and triple combination mutations were introduced, they exhibited an additive enhancement that further improved T cell activation while retaining a high degree of specificity.

Graft-Versus-Host-Disease Recruits Both Monocyte and Pre-Conventional Dendritic Cell-Derived Tissue Antigen Presenting Cells Independent Of CCR2 and CCR6

Graft-versus-host disease (GVHD) limits the broader application of allogeneic hematopoietic stem cell transplantation. In prior studies we defined roles for both host and donor-derived antigen presenting cells (APCs) in the activation of alloreactive donor T cells and in promotion of GVHD. While initial T cell activation in GVHD occurs predominantly in secondary lymphoid organs, we have consistently observed MHCII+ donor-derived APCs, including dendritic cells (DCs), in histopathologic GVHD lesions, frequently adjacent to infiltrating T cells, suggesting they have a role in local GVHD reactions. Donor-derived tissue APCs (t-APCs), including tissue-DCs (t-DCs) could activate donor T cells through indirect or cross-presentation of host antigens, produce chemokines that recruit other effectors, and elaborate inflammatory mediators or suppressors of inflammation. We first characterized t-DC subsets in the skin and bowel of GVHD-affected mice. 129 (H-2b) hosts were irradiated and reconstituted with B6 (H-2b) BM with or without CD4+ and CD8+ T cells to induce GVHD and analyzed mononuclear cells from skin and bowel approximately 4 weeks post transplant. In skin, both main dermal DC populations (CD11b+ and CD103+) were significantly increased in GVHD mice as compared to BM alone controls, though the ratios of CD11b+: CD103+ DCs were similar. In the bowel lamina propria, the ratios of CD11b+CD103- to CD11b+CD103+ were increased in GVHD mice in the colon but were similar to that in BM alone controls in the small bowel. We next studied the roles of CCR6 and CCR2 in the recruitment of donor-derived APCs to skin and bowel. We transplanted mice with CCR6-/- BM in competition with wild type (wt) BM and found that the contribution of each to skin and bowel APCs matched their contributions to myeloid hematopoiesis in BM, spleen and blood, indicating that CCR6 is not required. To study the role of CCR2 we first compared mice transplanted with either wt or CCR2-/- BM with wt T cells. Despite having a profound reduction in blood monocytes, all skin and bowel t-APC subsets were present in CCR2-/- recipients, indicating that CCR2 is not required for t-APC recruitment in contrast to its role in many other models of inflammation. However, CD103+ DCs were more prevalent relative to CD11b+ DCs, consistent with a pre-cDC origin. Despite monocytopenia, recipients of CCR2-/- BM developed clinical GVHD; histology data is being analyzed and will be presented. To better define the contributions of CCR2 to t-APC recruitment and to determine monocyte versus pre-cDC origin of t-DCs, we transplanted mice with CCR2-/- BM in competition with wt BM and compared ratios of BM and blood precursors (pre-cDCs and monocytes) to t-DC ratios. For CD103+ DCs, wt/KO ratios matched the ratios of general myeloid hematopoiesis and pre-cDCs, indicating a pre-cDC origin. For CD11b+CD103- DCs, the ratio of wt/KO matched that in blood monocytes. We further subsetted CD11b+ t-DCs based on the expression of Ly6C, MAR1, CD64 and CD24, used to differentiate pre-cDC from mono-derived DCs in other organs, and did not identify any population with wt/KO ratios that did not match that of the general CD11b+ DC population, suggesting that most if not all CD11b+ t-DCs are of monocyte origin. Experiments are underway examining the role of CX3CR1 in t-APC recruitment and these data will be presented. Disclosures: No relevant conflicts of interest to declare.

Two Photon Intravital Microscopy Reveals CD4+ T Cells Making Cognate Interactions With Tissue Dendritic Cells In Skin Graft-Versus-Host-Disease

Graft-versus-host disease (GVHD) limits the broader application of allogeneic hematopoietic stem cell transplantation. In prior studies we defined roles for both host and donor-derived antigen presenting cells (APCs) in the activation of alloreactive donor T cells and promotion of GVHD. While initial T cell activation in GVHD occurs predominantly in secondary lymphoid organs (SLO), we have consistently observed MHCII+ donor-derived tissue APCs (t-APCs), including tissue dendritic cells (t-DCs) in histopathologic GVHD lesions, frequently adjacent to infiltrating T cells. We hypothesize that productive interactions occur between donor APCs and T cells in situ in GVHD target tissues, which propagate disease locally. We could not use knockout or APC depletion approaches to study T cell: t-APCs interactions as they impact APCs systemically and might therefore affect T cell stimulation in SLO. We therefore utilized two-photon intravital microscopy to analyze interactions between fluorescent donor CD4+ T cells and t-DCs in skin. 129 (H-2b) hosts were irradiated and reconstituted with B6 (H-2b) CD11c-YFP transgenic (Tg) Bone Marrow (BM) with B6 RFP Tg CD4 cells and nonfluorescent B6 CD8 cells. We imaged ear skin in GVHD mice 4 weeks later. In general CD4 cells co-localized with DCs. We observed CD4+ T cells that were highly motile and only in transient contact with DCs and others that made stable contact with DCs. To determine how much TCR: MHCII interactions drive sustained CD4+ T cell: DC interactions and arrest CD4+ T cell motility, mice were imaged and then injected with an MHCII blocking antibody (Ab; Y3P) with continued imaging of the same regions. After injection, T cell mean speed significantly increased and the proportion of T cells in stable contact with DCs decreased, indicating that transient disruption of TCR: MHCII is sufficient to restore motility to some T cells. In a second approach to assess the specificity of CD4+ T cell: t-DC interactions we transplanted 129 mice with B6 RFP+ CD4 cells, nonfluorescent CD8 cells and a mix of CD11c-YFP MHCII-/- and RFP wt BM or a mix of CD11c-YFP wt and RFP Tg MHCII-/- BM. We are currently comparing the motility of CD4 cells that make contact with MHCII+ as compared to MHCII- DCs, with the prediction that contact times will be shorter with the latter. Our data suggest that CD4+ T cells make cognate interactions with t-DCs in skin and we hypothesize that these interactions promote GVHD locally. Because the graft-versus-leukemia effect occurs primarily in bone marrow and secondary lymphoid tissues, targeting of tissue-infiltrating APCs could represent a unique strategy to ameliorate GVHD while preserving GVL. Disclosures:No relevant conflicts of interest to declare.

Infusion of CD19-Directed and Multivirus Specific Cytotoxic T Lymphocytes After Allogeneic Hematopoietic Stem Cell Transplantation for B Cell Malignancies,

Abstract 4083Allogeneic hematopoietic stem cell transplant (HSCT) may increase long term disease free survival in patients with high risk B-cell malignancies. The delayed immune reconstitution associated with the procedure is, however, associated with significant morbidity and mortality from infections with cytomegalovirus (CMV), Epstein Barr virus (EBV), and adenoviruses (AdV). Disease relapse is another common cause of treatment failure. We reasoned that a single T cell platform mediating both antiviral and antileukemic activity could benefit these patients. We prepared cytotoxic T lymphocytes with specificities through their native receptors that were directed toward EBV, CMV and adenovirus; we then engineered these cells to express a chimeric antigen receptor (CAR) directed against CD19 (an antigen commonly present on B cell malignancies) and containing the co-stimulatory endodomain derived from CD28. We used donor derived antigen presenting cells (monocytes for the initial stimulation, EBV- lymphoblastoid cell lines, EBV-LCLs, for subsequent stimulations) that expressed adenovirus antigens and transgenic CMVpp65 following transduction with the adenoviral vector Ad5f35CMVpp65. After the third stimulation, multivirus-specific T cells were transduced with a retroviral vector encoding the CAR-CD19.28zeta transgene. Ten CTL cultures have been generated for clinical use, and contain a median of 71% (range 3–98%) CD8+ and 23% (range 0.1–92%) CD4+ cells, with a median of 14% (range 1–83%) CD45RA-/CD62L+ T cells. Transduction efficiency ranged from 20–55% (median 41%). These CTLs showed specific activity against B-cell leukemia/lymphoma targets in 51Cr release and/or IFNγ Elispot assays (mean specific lysis 48% at E:T ratio of 20:1, range 4–87%) and against CMV, EBV, and adenovirus peptide expressing target cells (mean specific lysis 28% at E:T ratio of 20:1, range 4–100%) but not recipient PHA blasts (<10%). Three patients (23/F, 58/M, and 56/M) with relapsed Ph+ALL (n=1) or B-CLL (n=2) have been treated with CAR-CD19.28z-CTLs. Patients received between 1.5 to 3 × 107CTLs/m2 without infusion related toxicity. In patient 1, who has the longest follow up, a three-fold increase in CMV–specific T cells was seen by 4 weeks post CTL infusion as detected by IFNγ Elispot assay. Responses to CMV peptides increased from 225 IFNγ-secreting cells (SFC/2.5 × 105 PBMC) pre-T cells to 782 SFC/2.5×105PBMC 6 weeks post CTLs and responses to CD19+ targets increased from 3 SFC/2.5×105 PBMC 1 week post T cells to 35 SFC/2.5×105 PBMC 6 weeks post CTLs. Detection of Transgenic T cells: CAR-CD19-transduced T cells have been detected by quantitative real-time PCR amplification in the peripheral blood of Patients 1, 2, and 3 up to 4, 1, and 0 weeks post infusion, respectively. Although already absent from peripheral blood, the transgenic cells were found at sites of disease. In Patient 1, T cells were detected in the bone marrow (44.8 copies/1000 ng of DNA) 9 weeks post T cell transfer. Patient 2 developed fever, diarrhea and hypotension 4 weeks post T cell therapy. Findings were consistent with ileitis at a known previous site of disease. Biopsy of the gut showed an abnormal absence of normal and malignant B cells, but significant levels of CAR-CD19.28z T cells by quantitative PCR (55.44 copies/1000 ng of DNA). Anti-Viral Activity of Transgenic Cells: No patient developed viral infection post CTL. Anti-Tumor Activity of Transgenic Cells: Patient 1 with Ph+ ALL had 4% blasts detectable in the peripheral blood at the time of the first infusion of CTLs which cleared within two weeks. She received a second infusion two months later. She became bcr abl negative but subsequently relapsed and died of progressive disease seven months post CTL. Patient 2 with CLL had resolution of lymphadenopathy within two weeks but following the disappearance of CTLs from peripheral blood showed evidence of disease progression two months post CTL. Patient 3 is too early in the course of therapy to evaluate for disease response. In summary, our initial findings show that immunotherapy with CTL targeting both CD19 through a chimeric receptor and multiple viral antigens through their native receptor can be well tolerated after allogeneic stem cell transplantation and may have both anti-viral and anti-tumor activity. Disclosures:No relevant conflicts of interest to declare.

Targeted ‘Off-the-Shelf' Tumor Immunotherapy Using Allogeneic T-Cell Precursors.

T cell deficiencies can occur in many settings, including aging, autoimmune and genetic disorders, hematological malignancies, infectious diseases, and exposure to cytotoxic/cytostatics agents. Notch-based culture systems can be utilized for ex vivo generation of large numbers of T lineage committed lymphoid precursor cells, and we recently reported that the adoptive transfer of T cell precursors significantly enhances T cell reconstitution and function after allogeneic T cell depleted HSCT. The aim of this study was to evaluate if allogeneic T cell precursors are safe and effective when used for adoptive transfer across MHC barriers in the absence of allogeneic HSCs to overcome radiation injury, enhance T cell function and improve anti-tumor activity in immunosuppressed recipients. We found that positive selection of adoptively transferred allogeneic (C57BL/6) precursor cells ± syngeneic (BALB/c) HSCs in irradiated hosts (BALB/c) is dependent on MHC molecules on non-hematopoietic host cells, resulting in the in vivo development of host-MHC restricted allogeneic T cells. We demonstrate that negative selection of these cells is mediated by donor-derived antigen presenting cells (APCs), resulting in host-tolerance. Adoptively transferred allogeneic T cell precursors significantly improved survival of BALB/c mice after irradiation (675 cGy) and enhanced anti-tumor activity against liquid (A20 lymphoma) and solid (renal cell carcinoma) tumors in syngeneic HSCT recipients. Furthermore, we demonstrate the feasibility of genetic engineering of antigen-specific T cell precursors, by transducing them to express a chimeric antigen receptor (CAR) targeting human CD19. Transduction efficiencies were routinely in the range of 50%–70%. Adoptive transfer of CAR-expressing T cell precursors resulted in the in vivo generation of high numbers of appropriately selected CAR-expressing T cells with significantly enhanced anti-tumor activity (compared with CAR-negative T cell precursors) against a CAR-sensitive tumor, but without any undesirable auto/alloreactivity. We conclude that adoptively transferred allogeneic T cell precursors develop into host-MHC restricted and host-tolerant T cells characterized by selection of a functional TCR repertoire even in a fully mismatched thymic epithelial MHC environment. This strategy overcomes important limitations of conventional adoptive T cell therapies: rejection, alloreactivity and impaired antigen recognition due to restriction to MHC disparate from the one expressed on APCs. The use of allogeneic instead of autologous cells eliminates the risk of contamination with residual malignant patient cells and allows the generation and storage of virtually unlimited quantities of precursor cells for ‘off-the-shelf' immunotherapy. This procedure has not only substantial logistic advantages, but it facilitates ex vivo manipulation, in particular genetic engineering, to generate antigen-specific or otherwise enhanced designer cells. Adoptive transfer of MHC mismatched and genetically enhanced T cell precursors therefore represents a promising novel strategy for targeted ‘off-the-shelf' immunotherapy in immunosuppressed patients.

Spontaneous Allograft Tolerance in B7-Deficient Mice Independent of Preexisting Endogenous CD4+CD25+ Regulatory T-Cells

Acute cardiac allograft rejection requires host, but not donor, expression of B7-1/B7-2 costimulatory molecules. However, acute cardiac rejection requires direct antigen presentation by donor-derived antigen presenting cells to CD4 T-cells and does not require indirect antigen presentation to CD4 T-cells. Given this discrepancy in the literature and that the consequence of allograft exposure in B7-deficient mice is unknown; the goal of the study was to examine the antidonor status of allografted B7-1/B7-2-deficient hosts. C57Bl/6 B7-1/B7-2-/- mice were grafted with heterotopic BALB/c hearts. Recipients bearing long-term surviving allografts were used to examine the status of antidonor reactivity in vitro and in vivo. Tolerance was examined in vivo through adoptive transfer of splenocytes from graft-bearing animals to secondary immune-deficient Rag-1-/- hosts bearing donor-type or third-party cardiac allografts and by regulatory T-cell depletion with anti-CD25 antibody. When transferred to B7-replete Rag-1-/- recipients, cells from naïve B7-1/B7-2-/- mice readily initiated cardiac allograft rejection. However, splenocytes transferred from long-term allograft acceptor B7-1/B7-2-/- hosts failed to reject donor-type hearts but acutely rejected third-party allografts. In addition, such cells did not reject (donorxthird-party) F1 allografts. Finally, in vivo depletion of regulatory T-cells did not prevent long-term acceptance. Results demonstrate that B7-deficient T-cells are capable of acute cardiac allograft rejection in a B7-replete environment. Importantly, results also show that B7-deficient hosts do not simply ignore cardiac allografts, but rather spontaneously develop transferable, donor-specific tolerance and linked suppression in vivo. Interestingly, this tolerant state does not require endogenous CD4+CD25+ regulatory T-cells.

Functional dichotomy of NK cells in organ transplantation

Evaluation of: Yu G, Xu X, Vu MD, Kilpatrick ED, Li XC. NK cells promote transplant tolerance by killing donor antigen-presenting cells. J. Exp. Med. 203, 1851–1858 (2006).Natural killer (NK) cells have the potential to display different functional activities after transplantation. The traditional view is that NK cells have the capacity to contribute to rejection by facilitating the activation/differentiation of leukocytes that destroy the graft. By contrast, in the article under review, a novel role for NK cells was identified in the setting of costimulation blockade where alloreactive NK cells of recipient origin were found to have the capacity to kill donor-derived antigen-presenting cells, thereby reducing T-cell priming and promoting long-term skin graft acceptance.

Donor age intensifies the early immune response after transplantation

Increasing donor age is associated with reduced graft function. We wondered if donor age may not only affect intrinsic function but also alter the immune response of the recipient. Kidneys from young and old F-344 rats (3 vs 18 months) were transplanted into bilaterally nephrectomized young Lewis recipients and compared with age-matched controls (follow-up: 6 months). Renal function and structural changes were assessed serially in both native kidneys and allografts. Host alloreactivity, graft-infiltrating cells, and their inflammatory products were determined at intervals to examine the correlation of immune response and donor age. Functional and structural deterioration had advanced significantly in older allografts compared with age-matched native controls, whereas differences between young allografts and native controls of similar age were only minor. Changes in grafts from elderly rats were associated with a more intense host immune response early post-transplant (up to 1 month) reflected by significantly higher numbers of peripheral T and B cells, increased T-cell alloreactivity and modified cytokine patterns associated with elevated frequencies of intragraft dendritic cells, B cells, and CD31+ cells. By 6 months, recipients of young donor grafts produced comparable or more intense alloantigen-specific immune responses. Older donor grafts elicit a stronger immune response in the early period after transplantation.

Nuclear Translocation of RelB within Host and Donor Antigen Presenting Cells Is Critical for the Initiation and Maintenance of Acute Graft-Versus-Host Disease.

Either donor or host antigen presenting cells (APC) are sufficient for the initiation of CD4 dependent graft versus host disease (GVHD). However the molecular transcription pathways within APC required for this effect are unknown. The NF-kB/Rel family member RelB is associated with dendritic cell (DC) maturation and is critical for the induction of potent APC function. DC from RelB−/− mice had markedly reduced levels of CD40 and to a lesser extent CD80/CD86 following in vitro activation. Following total body irradiation, the number of residual splenic DC with nuclear RelB was increased 5-fold relative to untreated mice. We therefore examined the role of RelB within donor and host APC in GVHD utilizing two well established bone marrow transplant models of CD4-dependant GVHD. To study the requirement of RelB within host APC we generated chimeric mice by transplanting wild-type (wt) or RelB−/− B6 bone marrow into irradiated wt B6 mice. Following immune reconstitution 4–6 months later, the number and frequency of DC (CD11chi and CD11cdimB220+) was equivalent in RelB−/− and RelB+/+ chimeras, although RelB−/− chimeras were specifically deficient in CD11chiCD4+ DC. Chimeras were subsequently transplanted with allogeneic Balb/c bone marrow and purified T cells. The absence of RelB within host APC significantly improved survival (survival day 60: 83% v 19%, P< .0001) and GVHD clinical scores were significantly reduced in RelB−/− chimeras for the first 4 weeks after transplant but subsequently rose to levels equivalent to those in surviving RelB+/+ chimeras. All RelB−/− and RelB+/+ chimeras that received syngeneic grafts survived without clinical evidence of GVHD. Sera from RelB−/− chimera recipients of allogeneic grafts contained reduced IFNg (117 ± 23 vs 253 ± 45 pg/ml; P< 0.02) and increased IL-5 (358 ± 105 vs 112 ± 20 pg/ml; P<0.05) compared to RelB+/+ chimera recipients (mean ± SE). Furthermore, CD4 T cells purified from the spleens of RelB−/− chimera recipients produced 2.6 fold more IL-4 (451 ± 31 vs 168 ± 17 pg/ml; P=0.01) than those from RelB+/+ chimera recipients. Taken together these data suggest the absence of nuclear RelB translocation within host APC abrogates GVHD and this is associated with the induction of donor Th2 differentiation. To study the role of RelB within donor APC we transplanted wt or RelB−/− B6 bone marrow and wt purified T cells into irradiated B6D2F1 recipients. In this model, GVHD severity was identical for the first 4 weeks after transplant but subsequently GVHD clinical scores in the recipients of RelB−/− donor APC returned toward levels seen in syngeneic recipients (clinical scores at day 49: 1.0 ± 0.6; n=6 vs 3.75 ± 0.4; n=6; RelB−/− vs RelB+/+P=0.01). This attenuation of acute GVHD in recipients of RelB−/− donor-derived APC was associated with the reconstitution of donor DC on day 21. These data suggest the inhibition of the nuclear RelB translocation within APC represents a potential new therapeutic target for the prevention of allograft rejection and GVHD.

Stem cell–mediated tolerance inducing strategies in organ transplantation

The scope of possible tools to modulate the recipients immune response towards tolerance induction basically includes deletional and non deletional mechanisms, which are currently targeted by various strategies including monoclonal antibodies, cytokine deviation, chimerism induction and the support of regulating T-cells. Here we summarize the main findings in the field derived from experimental animal studies and currently performed clinical studies. This review focuses to give a clinically relevant overview over relevant tolerance inducing concepts, taking into consideration risk profiles and clinical efficacy associated with specific immunosuppressive regiments currently applied in the clinical setting of transplantation.

TARGETING GENE THERAPY FOR TRANSPLANTATION WITH VENOM

TARGETING GENE THERAPY FOR TRANSPLANTATION WITH VENOM

Impact of Flt-3 ligand on donor-derived antigen presenting cells and alloimmune reactivity in heart graft recipients given adjuvant donor bone marrow

The influence of the haematopoietic growth factor Flt-3 ligand (FL) on the incidence and function of donor major histocompatibility complex (MHC) class II + cells in the lymphoid tissues of non-cytoablated recipients of heart allografts and donor bone marrow (BM) cells was investigated. C3H (H2 k) mice received a nonvascularized B10 (H2 b) heart allograft in the dorsal ear pinna, followed by an i.v. influsion of 50 × 10 6 donor BM cells. They were given FL (10 μg/day i.p., ×7 days), tacrolimus (2mg/kg/day i.p., ×13 days) or both agents immediately following heart transplantation (HTx) and were killed 10 or 21 days later. Their BM cells were propagated in vitro in granulocyte macrophage colony stimulating factor (GM-CSF) and IL-4 for 5 days to promote the growth of dendritic cells (DC). Donor DC were identified by immunocytochemical staining. Spleens were harvested, and donor (IA b+) cells enumerated by immunohistochemical analysis. Donor MHC class II DNA was detected in spleens and cultured BM-derived cells by reverse transcriptase-polymerase chain reaction (RT-PCR). A striking increase in donor MHC class II + cells was noted in both the spleen and BM of the BM + tacrolimus-treated group compared to either the BM alone, or BM + FL-treated groups. Addition of FL treatment to BM + tacrolimus led to a further increase in donor cells in spleen (three-fold at 10 days, and two-fold at 21 days) The increase in donor cells at 10 days was almost 140-fold compared to that with donor BM alone. PCR analysis at this time revealed enhanced donor DNA in the BM + FL + tacrolimus group compared to that in the BM + tacrolimus group. FL treatment augmented mixed leucocyte reactions (MLR) and cytotoxic T lymphocyte (CTL) activity of host spleen cell against donor alloantigens. These effects were reversed by tacrolimus administration. Histopathology of heart grafts from tacrolimus-treated animals at 10 and 21 days showed absence or substantial reduction in cellular infiltration, and the preservation of viable myocardium. By contrast, in untreated mice, or animals given BM or BM + FL alone, there was marked cellular infiltration, and features of accelerated rejection. Donor-derived DC could be propagated in vitro from the BM of heart transplant recipients given donor BM, especially from mice that also received tacrolimus ± FL. At day 21, donor-derived cells could only be propagated from the BM + FL + tacrolimus-treated group. These findings show that numbers of donor antigen presenting cells (APC) or their progenitors can be markedly

A crucial role of host CD80 and CD86 in rat cardiac xenograft rejection in mice.

Graft rejection can be initiated by two primary pathways of antigen presentation: (a) direct activation of host T cells by donor-derived antigen presenting cells (APC) and (b) indirect presentation of processed graft antigens by host APC. We investigated the differential roles for direct and indirect antigen presentation by preventing the CD28 costimulatory pathway with monoclonal antibodies to rat or mouse CD80 and CD86 in a rat-to-mouse cardiac transplantation model. Although the mouse anti-rat monoclonal antibodies to CD80 and CD86 did not significantly prolong the survival of rat cardiac xenografts in mice, the rat anti-mouse monoclonal antibodies to CD80 and CD86 did prolong the survival. Development of the anti-donor antibodies was inhibited, and the deposition of C3, IgM, and IgG on endothelium in the xenografts was mild in the anti-mouse CD80/CD86-treated mice. Infiltration of macrophages, neutrophils, and lymphocytes expressing perforin and interferon-gamma was decreased by the anti-mouse CD80/CD86 treatment. These findings suggest that the indirect antigen presentation, which is mediated by CD80 and CD86 pathway on host APC, plays a crucial role in concordant cardiac xenograft rejection.