Alloreactive CD4 Research Articles

In Vivo Expansion of Recipient DC Leads to Deletion of Donor Alloreactive CD8+ T Cells and Is an Alternative to Post-Transplant Cyclophosphamide for the Prevention of Graft-Versus-Host Disease after BMT

Allogeneic bone marrow transplantation (BMT) provides curative therapy for leukemia via immunological graft-versus-leukemia (GVL) effects. In practice, this must be balanced against graft-versus-host disease (GVHD) and opportunistic infection. We have investigated the role of recipient dendritic cells (DC) in the control of MHC class I restricted T cell responses after BMT. We first used the C3H.Sw → B6 miHA mismatched model of mouse BMT, with lethally irradiated B6.CD11c.DOG recipients (diphtheria toxin receptor, ovalbumin and GFP driven off the CD11c promoter) such that recipient DC can be deleted by diphtheria toxin, in combination with B6-derived MLL-AF9 induced primary acute myeloid leukemia to model clinical relapse. Depletion of recipient DC resulted in improved leukemic control (median survival 43 vs 31 days, P <0.001). The use of IRF8-/- BMT recipients (CD8α+ DC subset absent) confirmed that recipient CD8α+ DC were critical for regulating these GVL effects (median survival 43 vs 34 days, P = 0.0005). We confirmed that this accelerated relapse occurred as a consequence of recipient DC-induced donor T cell apoptosis resulting in a profound contraction of the donor T cell compartment. Using the same depletion strategies in an antigen-specific model (donor OT-I T cells, B6.CD11c.DOG x DBA/2 F1 recipients) we confirmed that recipient DC invoked effector donor CD8+ T cell hyperactivation, differentiation and apoptosis (Annexin V+ OT-I; 52.4% vs. 23.9% in DC replete vs. depleted recipients, P = 0.01).In light of the effects of recipient DC on antigen-specific donor T cell deletion, we next compared pre-transplant Flt-3L treatment (Pre-T Flt-3L; d -10 to d 0) to expand recipient CD8α+ DC (mean CD8α+ DC/spleen saline 0.14×106 vs Pre-T Flt-3L 23.6×106; P= 0.008) with post-transplant cyclophosphamide (PT-Cy at d 3,4), in a model of haploidentical BMT (B6 → B6D2F1). Consistent with our DC depletion models, Pre-T Flt-3L resulted in augmented antigen-specific donor T cell expansion 12 hours post-BMT, followed by deletion within 72 hrs (d 3 mean splenic OT-I: saline 15.94×106 vs Pre-T Flt-3L 0.01×106; P= 0.02). PT-Cy resulted in similar profound but alloantigen-independent polyclonal T cell deletion (d 7 mean splenic CD3+: saline 10.8×106 vs PT-Cy 0.8×106; P= 0.02). Pre-T Flt-3L offered better protection from GVHD than PT-Cy (median survival 36 vs 45 days, P= 0.02). In contrast, cyclosporine (CsA, administered to deliver clinically relevant serum levels of 200 - 300 ng/mL) did not result in major improvements in GVHD outcome (median survival 11 vs 13 days, P= 0.07). Both Pre-T Flt-3L and PT-Cy ablated GVL effects with relapse rates similar to corresponding TCD controls (Pre-T Flt3-L median time to relapse 11 days TCD vs 12 days BM+T; PT-Cy 22 days TCD vs 26 days BM+T). In contrast, GVL was maintained after CsA (with long-term survival seen in CsA and saline treated BM+T mice, median survival not reached; P >0.99). We confirmed that the effects with Pre-T Flt-3L were DC specific, and not related to the other populations by simultaneously deleting recipient DC (using diphtheria toxin) in Flt-3L treated recipients. This completely restored GVL effects and long-term survival to that observed in non-Flt-3L treated recipients (P >0.99). Importantly, despite effects on antigen-specific T cells, Pre-T Flt-3L facilitated the maintenance of third party CMV-specific donor T cells whereas PT-Cy did not (m38 tetramer+ cells 3 days post treatment; saline vs Pre-T Flt-3L P= 0.27; saline vs PT-Cy P= 0.028). These data highlight pre-transplant Flt-3L therapy as a potent alternative therapeutic strategy to PT-Cy to prevent GVHD, and moreover, that the effects of these strategies on attenuating GVL may have important clinical ramifications. DisclosuresLane:novartis: Consultancy; janssen: Consultancy, Research Funding.

Mechanisms of GVL-Resistance: T Cell Exhaustion and a Failure of Antigen Cross-Presentation

In MHC-matched allogeneic hematopoietic stem cell transplantation (alloSCT), minor histocompatibility antigens (miHAs) are the major targets of alloreactive CD8+ T cells that mediate the GVL effect. Despite the development of alloreactive CD8 cells in most if not all patients, relapsed leukemia is the greatest single cause of treatment failure. Why relapse occurs has been elusive. Previously we tested adoptive immunotherapy with bona fide CD8+ memory T cells (TM) from donors vaccinated against a single miHA expressed by leukemia cells. Vaccination against the Kb-restricted miHA H60 (LTFNYRNL) greatly augmented TM-mediated GVL against mouse chronic phase and blast crisis chronic myeloid leukemia (BC-CML). In TMH60 recipients, despite a rapid expansion of H60-reactive T cells (measured by tetramer staining and IFN-g production) and an impressive GVL effect, we consistently observed persistent leukemia and later death in a subset of mice. We used this system to test hypotheses on GVL failure as we could track and enumerate H60-reactive CD8 cells, the only cells that mediate GVL in this system, by tetramer staining, which is not possible in traditional polyclonal GVL models. We hypothesized that GVL resistance could be due to immune selection of GVL-resistant leukemia cells and/or an insufficient T cell response. However, we found no evidence of immune selection. H60+ leukemia cells harvested from mice with relapsed BC-CML after an initially effective TMH60 GVL response and H60+ leukemia cells from transplanted mice that did not receive donor T cells were similarly sensitive to in vivo killing by both H60-reactive T cells and by OT-1 effector T cells when leukemia cells were pulsed with SIINFEKL. An insufficient T cell response could have been due to insufficient presentation of H60, as H60 is largely restricted to host hematopoietic cells, which are eliminated by the transplant. Indeed, H60 presentation was inefficient as fresh congenically marked TMH60, transferred 14 days post initial transplantation with TMH60, proliferated poorly in mice with or without leukemia. However, infusion of an agonist anti-CD40 antibody, also on day 14, promoted the proliferation and GVL activity of fresh TMH60 but not by the TeffH60+ (H60-reactive CD8 effectors) derived from TMH60 infused on day 0. The activity of anti-CD40 was mostly abrogated when donor BM was CD40-/-. Hence, cross-presentation by donor-derived cells is limiting, but T cells that initially mediated GVL were also poorly responsive. Relative to progeny from TMH60 infused on day 14, these cells had higher levels of PD-1, TIM-3, and TIGIT and a lower level of IFN-g production; this was even more marked in mice with residual leukemia. The increased exhaustion in mice with residual leukemia required the leukemia cells to present H60, as it did not occur when leukemias were H60+ but Kb-/-. Using the H60+Kb-/- BC-CML we found little evidence for cross-presentation of leukemia-derived H60 indicating that T cell responses against leukemia-restricted antigens is inefficient. Consistent with this, the anti-CD40 effect promoted cross-presentation of recipient and not leukemia-derived H60. In vivo treatment with anti-PD-1, but not anti-TIM-3 mAb, greatly augmented TMH60 mediated GVL without causing clinical GVHD as the TMH60 only target host hematopoietic cells. Day 0 treatment with anti-CD40 also augmented TMH60 mediated GVL without an increase in GVHD; but in this case the activity was on host cells. Taken together, our data support both exhaustion and a lack of antigen presentation later post BMT as factors that contribute to an insufficient T cell response, thus leading to GVL resistance. DisclosuresNo relevant conflicts of interest to declare.

Regulatory Dendritic Cells Induced by Bendamustine Are Associated With Enhanced Flt3 Expression and Alloreactive T-Cell Death.

The growth factor Flt3 ligand (Flt3L) is central to dendritic cell (DC) homeostasis and development, controlling survival and expansion by binding to Flt3 receptor tyrosine kinase on the surface of DCs. In the context of hematopoietic cell transplantation, Flt3L has been found to suppress graft-versus-host disease (GvHD), specifically via host DCs. We previously reported that the pre-transplant conditioning regimen consisting of bendamustine (BEN) and total body irradiation (TBI) results in significantly reduced GvHD compared to cyclophosphamide (CY)+TBI. Pre-transplant BEN+TBI conditioning was also associated with greater Flt3 expression among host DCs and an accumulation of pre-cDC1s. Here, we demonstrate that exposure to BEN increases Flt3 expression on both murine bone marrow-derived DCs (BMDCs) and human monocyte-derived DCs (moDCs). BEN favors development of murine plasmacytoid DCs, pre-cDC1s, and cDC2s. While humans do not have an identifiable equivalent to murine pre-cDC1s, exposure to BEN resulted in decreased plasmacytoid DCs and increased cDC2s. BEN exposure and heightened Flt3 signaling are associated with a distinct regulatory phenotype, with increased PD-L1 expression and decreased ICOS-L expression. BMDCs exposed to BEN exhibit diminished pro-inflammatory cytokine response to LPS and induce robust proliferation of alloreactive T-cells. These proliferative alloreactive T-cells expressed greater levels of PD-1 and underwent increased programmed cell death as the concentration of BEN exposure increased. Alloreactive CD4+ T-cell death may be attributable to pre-cDC1s and provides a potential mechanism by which BEN+TBI conditioning limits GvHD and yields T-cells tolerant to host antigen.

IL-33 is a critical early activation and differentiation signal for alloreactive CD4+ T cells that enables their infiltration into GVHD target tissues

Abstract Graft vs host disease (GVHD) is a lethal complication after allogeneic stem cell transplantation (alloSCT), where donor T cells destroy host tissues. To create space for donor cells and prevent their rejection, alloSCT protocols use recipient conditioning that augments the stromal-derived cytokine interleukin (IL)-33 in the lymphoid organs (LO) and tissues. While we have established that IL-33 is a clinically-relevant signal in GVHD, mechanisms by which it contributes to alloimmunity are not well defined. Herein, we used IL-33 receptor, ST2, deficient B6 mice as T cell donors to elucidated IL-33-mediated mechanisms in the early activation and differentiation of alloreactive T cells. In our model, we transferred equal ratios of B6 Cd4-Cre x St2fl/fl (ST2ko) and Cd4-Cre (ST2wt) donor T cells into irradiated B6 and BALB/c recipients. Donor T cells in the LO and tissues were characterized at day 1–3, 5, and 7 post alloSCT. BALB/c recipients exhibited blunted early activation and proliferation of alloresponsive ST2ko CD4+ T cells compared to ST2wt CD4+ T cells in LO of the same recipient. ST2ko T cells in allogeneic recipients also failed to differentiation into Tbethi and CXCR3hi Th1 cells able to infiltrate into target tissues and cause GVHD. RNAseq analysis confirmed the need for IL-33-signaling for alloreactive Th1 generation as ST2KO CD4+ cells exhibited reduced IFNγ, Tbet, CXCR3, and instead expressed higher levels of Th2 and regulatory molecules (Foxp3, IL10, Gata3). IL-33 signaling was not required for the non-alloantigen driven lymphopenic expansion by CD4+ T cells in syngeneic recipients. These data establish a previously unappreciated and targetable role for IL-33 in effective alloAg-reactive donor Th1 generation after alloSCT.

A Role for Human Renal Tubular Epithelial Cells in Direct Allo-Recognition by CD4+ T-Cells and the Effect of Ischemia-Reperfusion.

Direct allorecognition is the earliest and most potent immune response against a kidney allograft. Currently, it is thought that passenger donor professional antigen-presenting cells (APCs) are responsible. Further, many studies support that graft ischemia-reperfusion injury increases the probability of acute rejection. We evaluated the possible role of primary human proximal renal tubular epithelial cells (RPTECs) in direct allorecognition by CD4+ T-cells and the effect of anoxia-reoxygenation. In cell culture, we detected that RPTECs express all the required molecules for CD4+ T-cell activation (HLA-DR, CD80, and ICAM-1). Anoxia-reoxygenation decreased HLA-DR and CD80 but increased ICAM-1. Following this, RPTECs were co-cultured with alloreactive CD4+ T-cells. In T-cells, zeta chain phosphorylation and c-Myc increased, indicating activation of T-cell receptor and co-stimulation signal transduction pathways, respectively. T-cell proliferation assessed with bromodeoxyuridine assay and with the marker Ki-67 increased. Previous culture of RPTECs under anoxia raised all the above parameters in T-cells. FOXP3 remained unaffected in all cases, signifying that proliferating T-cells were not differentiated towards a regulatory phenotype. Our results support that direct allorecognition may be mediated by RPTECs even in the absence of donor-derived professional APCs. Also, ischemia-reperfusion injury of the graft may enhance the above capacity of RPTECs, increasing the possibility of acute rejection.

Retinoic acid–responsive CD8 effector T cells are selectively increased in IL-23–rich tissue in gastrointestinal GVHD

AbstractGastrointestinal (GI) graft-versus-host disease (GVHD) is a major barrier in allogeneic hematopoietic stem cell transplantation (allo-HSCT). The metabolite retinoic acid (RA) potentiates GI-GVHD in mice via alloreactive T cells expressing the RA receptor-α (RARα), but the role of RA-responsive cells in human GI-GVHD remains undefined. Therefore, we used conventional and novel sequential immunostaining and flow cytometry to scrutinize RA-responsive T cells in tissues and blood of patients who had received allo-HSCT and to characterize the impact of RA on human T-cell alloresponses. Expression of RARα by human mononuclear cells was increased after exposure to RA. RARαhi mononuclear cells were increased in GI-GVHD tissue, contained more cellular RA-binding proteins, localized with tissue damage, and correlated with GVHD severity and mortality. By using a targeted candidate protein approach, we predicted the phenotype of RA-responsive T cells in the context of increased microenvironmental interleukin-23 (IL-23). Sequential immunostaining confirmed the presence of a population of RARαhi CD8 T cells with the predicted phenotype that coexpressed the effector T-cell transcription factor T-bet and the IL-23–specific receptor (IL-23R). These cells were increased in GI- but not skin-GVHD tissues and were also selectively expanded in the blood of patients with GI-GVHD. Finally, functional approaches demonstrated that RA predominantly increased alloreactive GI-tropic RARαhi CD8 effector T cells, including cells with the phenotype identified in vivo. IL-23–rich conditions potentiated this effect by selectively increasing β7 integrin expression on CD8 effector T cells and reducing CD4 T cells with a regulatory cell phenotype. In summary, we have identified a population of RA-responsive effector T cells with a distinctive phenotype that is selectively expanded in human GI-GVHD and that represents a potential new therapeutic target.

CD8+ T-Cell Repertoire in Human Leukocyte Antigen Class I-Mismatched Alloreactive Immune Response.

In transplantation, direct allorecognition is a complex interplay between T-cell receptors (TCR) and HLA molecules and their bound peptides expressed on antigen-presenting cells. In analogy to HLA mismatched hematopoietic stem cell transplantation (HSCT), the TCR CDR3β repertoires of alloreactive cytotoxic CD8+ responder T cells, defined by the cell surface expression of CD137 and triggered in vitro by HLA mismatched stimulating cells, were analyzed in different HLA class I mismatched combinations. The same HLA mismatched stimulatory cells induced very different repertoires in distinct but HLA identical responders. Likewise, stimulator cells derived from HLA identical donors activated CD8+ cells expressing very different repertoires in the same mismatched responder. To mimic in vivo inflammation, expression of HLA class l antigens was upregulated in vitro on stimulating cells by the inflammatory cytokines TNFα and IFNβ. The repertoires differed whether the same responder cells were stimulated with cells treated or not with both cytokines. In conclusion, the selection and expansion of alloreactive cytotoxic T-cell clonotypes expressing a very diverse repertoire is observed repeatedly despite controlling for HLA disparities and is significantly influenced by the inflammatory status. This makes prediction of alloreactive T-cell repertoires a major challenge in HLA mismatched HSCT.

Glycolytic metabolism of pathogenic T cells enables early detection of GVHD by 13C-MRI

AbstractGraft-versus-host disease (GVHD) is a prominent barrier to allogeneic hematopoietic stem cell transplantation (AHSCT). Definitive diagnosis of GVHD is invasive, and biopsies of involved tissues pose a high risk of bleeding and infection. T cells are central to GVHD pathogenesis, and our previous studies in a chronic GVHD mouse model showed that alloreactive CD4+ T cells traffic to the target organs ahead of overt symptoms. Because increased glycolysis is an early feature of T-cell activation, we hypothesized that in vivo metabolic imaging of glycolysis would allow noninvasive detection of liver GVHD as activated CD4+ T cells traffic into the organ. Indeed, hyperpolarized 13C-pyruvate magnetic resonance imaging detected high rates of conversion of pyruvate to lactate in the liver ahead of animals becoming symptomatic, but not during subsequent overt chronic GVHD. Concomitantly, CD4+ T effector memory cells, the predominant pathogenic CD4+ T-cell subset, were confirmed to be highly glycolytic by transcriptomic, protein, metabolite, and ex vivo metabolic activity analyses. Preliminary data from single-cell sequencing of circulating T cells in patients undergoing AHSCT also suggested that increased glycolysis may be a feature of incipient acute GVHD. Metabolic imaging is being increasingly used in the clinic and may be useful in the post-AHSCT setting for noninvasive early detection of GVHD.

IL-10 and TGF-β induce alloreactive CD4+CD25– T cells to acquire regulatory cell function

AbstractWe previously reported that interleukin-10 (IL-10) and transforming growth factor (TGF)–β treatment of primary mixed lymphocyte reaction (MLR) cultures resulted in secondary alloantigen-specific hyporesponsiveness and protection from graft-versus-host disease (GVHD) lethality. Here, we report that CD4+ T cells recovered from the IL-10– and TGF-β–treated primary MLR cultures have immunoregulatory function. Tolerized cells significantly inhibited proliferation of naive alloreactive CD4+ T cells in a primary MLR. Inhibition of the naive alloresponse was observed with as few as 1 tolerized cell to 10 naive responder cells. Tolerized cells were able to significantly reduce GVHD lethality when injected with naive alloreactive CD4+ T cells into major histocombatibility class (MHC) II disparate recipients. Rigorous CD25 depletion of the primary MLR had no effect on generation of a regulatory capacity, suggesting that the regulatory cells likely originated from CD4+CD25– T cells. Immune suppression was mediated independently of IL-10 and TGF-β production, as neutralizing antibodies for IL-10, IL-10R, and TGF-β were unable to revert suppression, and IL-10– deficient CD4+ T cells were able to mediate in vitro and in vivo suppression. The generation of immunoregulatory cells from a CD4+CD25– population during tolerization with IL-10 and TGF-β provides an additional mechanism to prevent GVHD lethality by T cells that may escape full tolerance induction.

Back cover story: Eur. J. Immunol. 12'20

European Journal of ImmunologyVolume 50, Issue 12 Back CoverFree Access Back cover story: Eur. J. Immunol. 12'20 First published: 11 December 2020 https://doi.org/10.1002/eji.202070128AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinked InRedditWechat Abstract Our back cover features an immunofluorescence-staining showing recipient origin of DNGR-1+ dendritic cells (DCs) with positivity for H-2b (H-2b+ DNGR-1+ DC, in violet, nuclei in blue) in allografts (BALB/c donor hearts) at day 5 after transplantation into wild-type C57BL/6 recipients. The image is taken from Balam et al. (pp. 2041–2054), where the authors present that cross-presentation of dead-cell-associated antigens by DNGR-1+ DCs contributes to chronic allograft rejection in mice. The study provides novel insights into the mechanism by which DNGR-1-dependent alloantigen cross-presentation by DNGR-1+ DCs induces alloreactive CD8+ cells that in a downstream cascade induce chronic allograft rejection and fibrosis. Antibody against DNGR-1 can block this process and prevent chronic allograft rejection and fibrosis. The original image was digitally modified for the cover. Volume50, Issue12December 2020 RelatedInformation

A Human-Mouse Hybrid HLA-A2-IG Dimer Inhibits the Proliferation of CD4+T Cells In Vitro

Currently, general immunosuppressive drugs are used to maintain tolerance to allografts. However, these drugs have a major drawback of rendering the patient susceptible to infections and other side effects like malignancy and drug related toxicities with an overall rejection of the organ at some point. Previous studies have shown that MHC-Ig dimers may suppress alloresponsive T cells in a donor specific manner in vitro. This work aimed to answer the question as to whether these dimers will surmount rejection through the direct mechanism of allorecognition by suppressing alloreactive CD8+ T cells. To do this, we first identified two mice models with a single mismatch at the MHC loci. We found and procured white albino NOD mice which happened to be transgenic for HLA-A2 and HLA-A24 molecules. We then constructed a human-mouse hybrid HLA-A2-Ig dimer by overlap-PCR to join parts of two different already cloned plasmids to form the full length HLA-A2β2α1α2murineα3 insert which was then cloned to pcDNA3.1 to form pcDNA3.1HLA-A2β2α1α2murineα3. The IgG2bFc region was added by restriction digestion and ligation to form the plasmid pcDNA3.1HLA-A2β2α1α2murineα3IgG2bFc. Sequencing was done and confirmed that the construction and cloning were successful. The plasmid pcDNA3.1HLA-A2β2α1α2murineα3IgG2bFc was then transfected by electroporation to J558L cells. Screening was done using G418 for 4 weeks in cell culture. We purified the dimer by affinity chromatography and then used ELISA to confirm expression of the dimer. The purified dimer was then used in 1-way MLC experiments where responder cells were mice cells expressing HLA-A24 molecules while stimulator cells were mice cells expressing HLA-A2 molecules. Cell samples were gated on anti-mouse CD3-PE/CY7, anti-mouse CD4-PE, and anti-mouse CD8-APC/CY7. Cell proliferation was analysed using CFSE. Our results showed that the proliferation of CD4+ T cells was inhibited in the presence of the dimer. This work is crucial for subsequent studies aiming to search for induction of donor specific tolerance.

LYG1 Deficiency Attenuates the Severity of Acute Graft-Versus-Host Disease Via Skewing T Cells Polarization Towards Treg Cells

Acute graft-versus-host disease (aGVHD) is a lethal complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). As a complex immunopathology, aGVHD depends on the recognition of host antigens by donor T cells and induces augmented response of alloreactive T cells. Despite considerable achievements in the treatment of aGVHD, it remains a major clinical problem for the patients undergoing allo-HSCT. Therefore, it is necessary to further illustrate new mechanisms and develop novel therapeutic strategies of aGVHD. Previously we reported LYG1 (Lysozyme G-like 1) as a novel classical secretory protein promoted antitumor function of T cell. In this study, the role of LYG1 in aGVHD was investigated. Firstly, we examined whether LYG1 affected the alloreactivity of CD4+ T cells in vitro by MLR assay and discovered that LYG1 deficiency reduced the activation of CD4+ T cells and Th1 ratio, but increased Treg ratio. Then we confirmed these observations using a major MHC mismatched aGVHD model by transferring T cells sorting from WT B6 or Lyg1-/- mice with bone marrow cells from WT B6 mice into lethally irradiated BALB/c mice. The alloreactive CD4+ T cells and the proportions of Th1 cells decreased whereas the proportions of Treg cells increased in spleens and livers in mice receiving Lyg1-/- T cells. LYG1-deficient T cells attenuated aGVHD severity, inhibited the expression of CXCL9 and CXCL10 and restrained CD4+ T cells infiltrating in livers. Furthermore, administration of recombinant LYG1 protein intraperitoneally aggravated aGVHD by promoting IFN-γ production. More importantly, LYG1 deficiency did not affect GVT (graft-versus-tumor) effects. In summary, we demonstrate LYG1 regulates aGVHD via modulating the alloreactivity of CD4+ T cells and differentiation of Th1/Treg cells. Our study indicates that LYG1 may be a novel target in aGVHD by mitigating aGVHD without impairing GVT function. The therapeutic effect of targeting LYG1 is required in future investigations. Funding This study was supported by grant from The National Natural Science Foundation of China (NSFC) (Grant Number 81600144) and grant from Research Foundation of Peking University First Hospital. Disclosures No relevant conflicts of interest to declare.

CTLA-4-IG PROLONGS GRAFT SURVIVAL SPECIFICALLY IN YOUNG BUT NOT IN OLD RECIPIENTS

Introduction: The proportion of older transplant recipients has increased dramatically. Age-specific effects of established immunosuppressants are poorly understood. Here, we assessed the impact of CTLA4-Ig, a fusion protein blocking costimulatory signaling between APCs and T cells through CD28. We hypothesized that the immunosuppressive capacity of CTLA4-Ig will depend on an age-specific expression of CD28 on alloreactive T-cells. Methods: Heart and skin grafts from young DBA2/J mice were transplanted into either young or old (2-3 and 18 mths, resp.) allogeneic C57BL/6 recipients treated with CTLA4-Ig or PBS; T-cell frequencies and subsets in addition to cytokine expression were assessed by flow cytometry and ELISA; expression of CD28 was assessed on young and old T-cell subsets. To delineate the age-specific immunosuppressive potential of CTL4-Ig, T-cells were transfected with a lentiviral vector inducing CD28 expression; transfected T-cells were subsequently transferred into young RAG2-/- mice receiving a fully mismatched skin graft. Results: Treatment with CTLA4-Ig prolonged skin graft survival only in young (p<0.01) but not in old recipients (p=0.33). Age-specific immunosuppressive effects of CTLA4-Ig were also confirmed in a SOT model: cardiac allografts in young mice treated with CTLA4-Ig survived indefinitely. In contrast, 80% of old recipients treated with CTLA4-Ig had lost their graft by 100 days. CTLA4-Ig reduced CD4+ central-memory and effector-memory T cells and diminished systemic IFN-γ levels only in young (p<0.05), but not old recipients (p=0.8). These differences correlated with a reduced expression of CD28 on antigen-experienced CD4+ T cells in old mice (p<0.001). Interestingly, Tregs displayed an increased expression of CD28 with age making them more prone to be targeted by CTLA4-Ig. Strikingly, adoptive transfer of old CD4+ T cells that were transfected with a lentiviral vector augmenting the expression of CD28 accelerated the rejection of allogeneic skin grafts in young RAG2-/- recipient mice (p<0.01 vs. controls animals that received adoptively transferred, non-transfected old T cells) demonstrating the cardinal role of CD28 expression on alloreactive CD4+ T cells. Conclusion: Immunosuppressive effects of CTLA4-Ig showed distinct age-dependent effects in both skin and cardiac transplantation linked to a reduced CD28 expression of alloreactive old T-cells in parallel to an augmented CD28 expression on old T-regs. Transfection experiments confirmed the critical role of CD28 in mounting robust alloimmune responses. These findings carry relevant clinical consequences.

Cross-presentation of dead-cell-associated antigens by DNGR-1+ dendritic cells contributes to chronic allograft rejection in mice.

The purpose of this study was to elucidate whether DC NK lectin group receptor-1 (DNGR-1)-dependent cross-presentation of dead-cell-associated antigens occurs after transplantation and contributes to CD8+ Tcell responses, chronic allograft rejection (CAR), and fibrosis. BALB/c or C57BL/6 hearts were heterotopically transplanted into WT, Clec9a-/- , or Batf3-/- recipient C57BL/6 mice. Allografts were analyzed for cell infiltration, CD8+ Tcell activation, fibrogenesis, and CAR using immunohistochemistry, Western blot, qRT2 -PCR, and flow cytometry. Allografts displayed infiltration by recipient DNGR-1+ DCs, signs of CAR, and fibrosis. Allografts in Clec9a-/- recipients showed reduced CAR (p<0.0001), fibrosis (P = 0.0137), CD8+ cell infiltration (P<0.0001), and effector cytokine levels compared to WT recipients. Batf3-deficiency greatly reduced DNGR-1+ DC-infiltration, CAR (P<0.0001), and fibrosis (P = 0.0382). CD8 cells infiltrating allografts of cytochrome C treated recipients, showed reduced production of CD8 effector cytokines (P<0.05). Further, alloreactive CD8+ Tcell response in indirect pathway IFN-γ ELISPOT was reduced in Clec9a-/- recipient mice (P = 0.0283). Blockade of DNGR-1 by antibody, similar to genetic elimination of the receptor, reduced CAR (P = 0.0003), fibrosis (P = 0.0273), infiltration of CD8+ cells (p = 0.0006), and effector cytokine levels. DNGR-1-dependent alloantigen cross-presentation by DNGR-1+ DCs induces alloreactive CD8+ cells that induce CAR and fibrosis. Antibody against DNGR-1 can block this process and prevent CAR and fibrosis.

Single-cell transcriptomics of alloreactive CD4+ T cells over time reveals divergent fates during gut graft-versus-host disease.

Acute gastrointestinal (GI) graft-versus-host disease (GVHD) is a primary determinant of mortality after allogeneic hematopoietic stem cell transplantation (alloSCT). The condition is mediated by alloreactive donor CD4+ T cells that differentiate into pathogenic subsets expressing IFN-γ, IL-17A, or GM-CSF and is regulated by subsets expressing IL-10 and/or Foxp3. Developmental relationships between Th cell states during priming in mesenteric lymph nodes (mLNs) and effector function in the GI tract remain undefined at genome scale. We applied scRNA-Seq and computational modeling to a mouse model of donor DC-mediated GVHD exacerbation, creating an atlas of putative CD4+ T cell differentiation pathways in vivo. Computational trajectory inference suggested emergence of pathogenic and regulatory states along a single developmental trajectory in mLNs. Importantly, we inferred an unexpected second trajectory, categorized by little proliferation or cytokine expression, reduced glycolysis, and high tcf7 expression. TCF1hi cells upregulated α4β7 before gut migration and failed to express cytokines. These cells exhibited recall potential and plasticity following secondary transplantation, including cytokine or Foxp3 expression, but reduced T cell factor 1 (TCF1). Thus, scRNA-Seq suggested divergence of alloreactive CD4+ T cells into quiescent and effector states during gut GVHD exacerbation by donor DC, reflecting putative heterogeneous priming in vivo. These findings, which are potentially the first at a single-cell level during GVHD over time, may assist in examination of T cell differentiation in patients undergoing alloSCT.

Complement-activated interferon-γ-primed human endothelium transpresents interleukin-15 to CD8+ T cells.

Alloantibodies in presensitized transplant candidates deposit complement membrane attack complexes (MACs) on graft endothelial cells (ECs), increasing risk of CD8+ T cell-mediated acute rejection. We recently showed that human ECs endocytose MACs into Rab5+ endosomes, creating a signaling platform that stabilizes NF-κB-inducing kinase (NIK) protein. Endosomal NIK activates both noncanonical NF-κB signaling to synthesize pro-IL-1β and an NLRP3 inflammasome to process and secrete active IL-1β. IL-1β activates ECs, increasing recruitment and activation of alloreactive effector memory CD4+ T (Tem) cells. Here, we report that IFN-γ priming induced nuclear expression of IL-15/IL-15Rα complexes in cultured human ECs and that MAC-induced IL-1β stimulated translocation of IL-15/IL-15Rα complexes to the EC surface in a canonical NF-κB-dependent process in which IL-15/IL-15Rα transpresentation increased activation and maturation of alloreactive CD8+ Tem cells. Blocking NLRP3 inflammasome assembly, IL-1 receptor, or IL-15 on ECs inhibited the augmented CD8+ Tem cell responses, indicating that this pathway is not redundant. Adoptively transferred alloantibody and mouse complement deposition induced IL-15/IL-15Rα expression by human ECs lining human coronary artery grafts in immunodeficient mice, and enhanced intimal CD8+ T cell infiltration, which was markedly reduced by inflammasome inhibition, linking alloantibody to acute rejection. Inhibiting MAC signaling may similarly limit other complement-mediated pathologies.

Allogeneic ADSCs Induce the Production of Alloreactive Memory-CD8 T Cells through HLA-ABC Antigens.

We investigated the immunogenicity of allogeneic human adipose-derived mesenchymal stem cells (ADSCs) through the production of alloreactive-CD8 T and -memory CD8 T cells, based on their human leukocyte antigen (HLA) expression. In surface antigen analysis, ADSCs do not express co-stimulatory molecules, but expresses HLA-ABC, which is further increased by exposure to the pro-inflammatory cytokines as well as IFN-γ alone. For immunogenicity analysis, allogeneic ADSCs cultured in xenofree medium (XF-ADSCs) were incubated with the recipient immune cells for allogeneic–antigen stimulation. As a result, XF-ADSCs induced IFN-γ and IL-17A release by alloreactive-CD8 T cells and the production of alloreactive-CD8 T cell through a direct pathway, although they have immunomodulatory activity. In the analysis of alloreactive memory CD8 T cells, XF-ADSCs also significantly induced the production of CFSE-low-CD8 TEM and -CD8 TCM cells. However, HLA-blocking antibodies significantly inhibited the production of CFSE-low memory-CD8 T cells, indicating that HLAs are the main antigens responsible for the development of allogeneic ADSCs’ immunogenicity. These results suggested that HLA surface antigens expressed in allogeneic MSCs should be solved in order to address concerns related to the immunogenicity problem.

The Role of PPARd in Alloreactive CD8 T cell Metabolism

Abstract Graft-versus-host disease (GVHD) is the most common cause of non-relapse mortality following allogeneic hematopoietic stem cell transplantation. Using a pre-clinical model of GVHD, previous work has found that CD8 T cells recovered on day 7 post-transplant upregulate the transcription factor, peroxisome proliferator activated receptor delta (PPARd). While PPARd has been shown to be a key regulator of fatty acid oxidation (FAO) in other tissues, it’s role in T cells is not well studied. We hypothesize that PPARd drives FAO in alloreactive CD8 T cells and that CD8 T cells lacking PPARd will be unable to oxidize fat and thus unable to cause GVHD. To investigate this hypothesis, ex vivo FAO was measured in day 7 WT versus PPARd KO CD8 T cells by quantitating conversion of 3H-palmitate to 3H2O. Production of 3H2O decreased by 50% in KO CD8 T cells, suggesting an inability to fully oxidize fat. To understand the mechanism underlying this decrease, we performed RNA sequencing and identified thirty genes that were differentially expressed in WT versus KO CD8s including 8 genes related to FAO. Strikingly, despite a decreased ability to oxidize fat, PPARd KO CD8 T cells were recovered in equal numbers on day 7 post transplantation, suggesting that CD8 T cells lacking PPARd adopt alternative metabolic pathways to generate sufficient energy for in vivo proliferation and activation. Ultimately, our analyses indicate that PPARd is required for FAO in alloreactive CD8 T cells, but this process is dispensable for short term proliferation and/or survival of CD8 T cells during GVHD. Future directions will identify compensatory metabolic pathways utilized by PPARd KO T cells through a combination of in vitro nutrient drop out experiments and metabolomic flux analyses.

MTOR Inhibitor Therapy Diminishes Circulating CD8+ CD28- Effector Memory T Cells and Improves Allograft Inflammation in Belatacept-refractory Renal Allograft Rejection.

Renal allograft rejection is more frequent under belatacept-based, compared with tacrolimus-based, immunosuppression. We studied kidney transplant recipients experiencing rejection under belatacept-based early corticosteroid withdrawal following T-cell-depleting induction in a recent randomized trial (Belatacept-based Early Steroid Withdrawal Trial, clinicaltrials.gov NCT01729494) to determine mechanisms of rejection and treatment. Peripheral mononuclear cells, serum creatinine levels, and renal biopsies were collected from 8 patients undergoing belatacept-refractory rejection (BRR). We used flow cytometry, histology, and immunofluorescence to characterize CD8 effector memory T cell (TEM) populations in the periphery and graft before and after mammalian target of rapamycin (mTOR) inhibition. Here, we found that patients with BRR did not respond to standard antirejection therapy and had a substantial increase in alloreactive CD8 T cells with a CD28/DR/CD38/CD45RO TEM. These cells had increased activation of the mTOR pathway, as assessed by phosphorylated ribosomal protein S6 expression. Notably, everolimus (an mTOR inhibitor) treatment of patients with BRR halted the in vivo proliferation of TEM cells and their ex vivo alloreactivity and resulted in their significant reduction in the peripheral blood. The frequency of circulating FoxP3 regulatory T cells was not altered. Importantly, everolimus led to rapid resolution of rejection as confirmed by histology. Thus, while prior work has shown that concomitant belatacept + mTOR inhibitor therapy is effective for maintenance immunosuppression, our preliminary data suggest that everolimus may provide an available means for effecting "rescue" therapy for rejections occurring under belatacept that are refractory to traditional antirejection therapy with corticosteroids and polyclonal antilymphocyte globulin.

Deletion of fetal-specific maternal CD4+ T cells during human pregnancy

Abstract Maternal T cell responses to fetal alloantigens are thought to play a critical role in facilitating successful pregnancy, but mechanisms of maternal-fetal tolerance remain largely unclear. Studies in murine models suggest that deletion of fetus-specific CD4+ T cells may contribute to tolerance; however, the role of deletion in human pregnancy is unknown. Here we use bulk TCR sequencing to assess changes in the maternal CD4+ T cell repertoire over the course of human pregnancy. Peripheral blood was collected from four primigravid women during the first and third trimesters, and a single peripheral blood sample was taken from their respective partners. Maternal CD4+ T cells were isolated using FACS, and TCR sequencing was performed on genomic DNA using the ImmunoSeqTM assay. The maternal alloreactive repertoire was defined using mixed lymphocyte reactions (MLRs) in which irradiated paternal PBMCs were used to stimulate maternal blood. An average of 32,077 (SD=9,609) unique CD4+ T cell clones/subject were identified at each timepoint. Within the small cohort examined in this study, we observed a notable increase in repertoire clonality in two of four women (average change in clonality=0.0045 vs. 0.0003). The two subjects that had an increase in CD4 clonality had a significant reduction in the number of alloreactive CD4 clones detected in the third trimester relative to the first whereas this number was effectively unchanged in the other two subjects (first:third=1:0.1±0.02 vs. 1:1.0±0.1). These findings suggest that substantial deletion of CD4+ alloreactive clones occurs in some women over the course of gestation. Deletion of alloreactive T cell clones may thus represent one mechanism of maternal-fetal tolerance in human pregnancy.