B6 Skin Research Articles

Early graft-infiltrating lymphocytes are not associated with graft rejection in a mouse model of skin transplantation.

Graft-infiltrating lymphocytes (GILs) play an important role in promoting rejection after organ transplantation. We recently reported that GILs that accumulated up to 3 days post-transplantation did not promote rejection, whereas GILs present 3-5 days post-transplantation promoted rejection in a mouse heart transplantation model. However, the immunological behaviour of GILs in murine skin transplantation remains unclear. GILs were isolated on days 3, 5 or 7 post-transplantation from C57BL/6 (B6) allogeneic skin grafts transplanted onto BALB/c mice. BALB/c Rag2-/- γc-/- mice (BRGs) underwent B6 skin graft transplantation 10 weeks after adoptive transfer of day 3, 5, or 7 GILs. BRGs reconstituted with day 5 or 7 GILs completely rejected B6 grafts. However, when B6 grafts harvested from recipient BALB/c mice on day 5 or 7 were re-transplanted into BRGs, half of the re-transplanted day 5 grafts established long-term survival, although all re-transplanted day 7 grafts were rejected. BRGs reconstituted with day 3 GILs did not reject B6 grafts. Consistently, re-transplantation using day 3 skin grafts resulted in no rejection. Administration of anti-CD25 antibodies did not prevent the phenomenon observed for the day 3 skin grafts. Furthermore, BRGs reconstituted with splenocytes from naïve BALB/c mice immediately rejected the naïve B6 skin grafts and the re-transplanted day 3 B6 grafts, suggesting that day 3 GILs were unable to induce allograft rejection during the rejection process. In conclusion, the immunological role of GILs depends on the time since transplantation. Day 3 GILs had neither protective nor alloreactive effects in the skin transplant model.

Immune checkpoint VISTA regulates DNA sensing and repair pathways that contribute to type I interferon (IFN-I) production in the skin

Abstract Skin sterile injury by ultraviolet (UV) light stimulates production of type I interferons. While in healthy skin IFN-I response to UV returns to baseline over time, the IFN-I signature persists in photosensitive diseases like lupus. The pathways that downregulate IFN-I production in healthy skin, and fail to do so in lupus skin, are unknown. Previous studies showing that activating VISTA suppresses the IFN-I signature in monocytes suggest a role for this immune checkpoint in the regulation of the IFN-I response. Indeed, we showed that the skin IFN-I signature is elevated in mice globally deficient in VISTA (Vsir−/−), as well as in mice with a conditional deletion of VISTA in keratinocytes. Having confirmed its expression in keratinocytes, we tested if VISTA regulates UV-triggered IFN-I production in the skin. Expression analysis of IFN-I stimulated genes in skin biopsies collected prior to and 3h after a single dose of UVB, revealed ~10-fold higher IFN-I score in Vsir−/− vs. B6 skin after UV. RNAseq studies showed DNA sensors, ZBP1, cGAS, and IFI204 were upregulated, while nucleotide excision repair genes, Polɛ and DNA Ligase1, were suppressed in Vsir−/− skin, both at baseline and 3h after UV. Moreover, Polɛ protein levels were lower in Vsir−/− skin by immunoblot. Relevant to its role in keratinocytes, IFN-k protein levels were higher in VISTA-deficient epidermal keratinocytes ex vivo, accompanied by elevated mRNA levels of ZBP1 and IFI204 before and after UV. Together, this study identifies VISTA as a novel regulator of IFN-I production, by modulating DNA damage repair and sensing. The keratinocyte-intrinsic role of VISTA is therapeutically relevant as lupus keratinocytes express high IFN-k and are direct targets of UV-induced DNA damage. 1R21AR079661 – 01, Hitchcock Foundation, DOD lupus award

Research Highlights

Ablation of Transcription Factor IRF4 Promotes Transplant Acceptance by Driving Allogenic CD4+ T Cell Dysfunction Jie Wu, Hedong Zhang, Xiamomin Shi, et al. Immunity. 2017;47(6):1114–1128.e6. The transcriptional programs regulating allogenic T-cell responses have not been adequately documented thus far. Here, the authors used a knockout mouse model to demonstrate the importance of interferon regulatory factor 4 (IRF4) in controlling graft rejection. IRF4 is a key transcriptional factor running T-cell responses during and after transplantation. The authors performed several experiments on IRF4 deficient (IRF4−/−) as well as wild type mice by transplanting them with mismatched heart allografts. First, they demonstrated that graft-infiltrating T cells expressed a consistently elevated mean fluorescence intensity of IRF4. Notably, they were able to show that IRF4−/− mice did not reject their allografts. Moreover, all IRF−/− mice survived the observation period of 100 days with intact myocytes and only minimal cellular infiltrates into the graft. The authors then went on transferring either 2 million wild type CD4+ or CD8+ T cells or 20 million IRF4−/− CD4+ or CD8+ T cells into IRF4−/− mice and found that the adaptive transfer of unmanipulated T cells facilitated rejection, whereas the transfer of IRF−/− T cells did not. Furthermore, IRF4−/− mice failed to show increased T cells in their spleen after transplantation, indicating that expansion of alloreactive T cells in IRF4−/− was impaired. Mechanistically, the authors demonstrated the critical role of PD-1 expression by alloantigen-specific T cells for the long-term acceptance of cardiac allografts in IRF−/− mice. Finally, the authors showed that trametinib, a MEK1/2 inhibitor most potently reduced IRF4 expression in activated T cells, inhibited Th1 and Th17 cell differentiation and prolonged heart allograft survival. This intriguing data show the critical role of IRF4 expression on T cells in alloimmunity and indicate the translational potential of trametinib to in improving transplant outcomes. Cutting Edge: Allograft Rejection Is Associated With Weak T-Cell Responses to Many Different Graft Leukocyte-Derived Peptides Burrack AL, Malhotra D, Dileepan T, Osum KC, Swanson LA, Fife BT, Jenkins MK. J Immunol. 2018; 200: 477–482. Allografts are rejected by T lymphocytes that recognize foreign MHC class I and II antigens. The robustness of this T-cell response, however, is not understood. Previous concepts have attributed the rejection process largely on the frequency of T cells that recognize either allogeneic MHC molecules alone or MHC plus-bound peptides. The authors explored the specificity of directly alloreactive T cells, using a peptide-MHC II tetramer-based approach. Tail skin from H2-DM- and CD74-deficient mice were grafted onto the flanks of recipient laboratory-bred strain house mouse. These grafts were rejected after 10 to 12 days. Spleen, lymph node, or blood cells were harvested and stained 7 to 14 days after transplantation. The total number of tetramer-positive cells in the enriched fraction from each mouse was determined, using AccuCheck counting beads. Differences in T-cell expansion and differentiations after transplantation between and across groups were established. CD4+ cells were identified as viable lymphocyte-sized single cells that expressed CD3 or CD90.2. Graft-responsive CD4 T cells expanded an average of 28-fold after transplantation. Mice transplanted with B6 skin also demonstrated a clonal T-cell expansion, indicating that this phenomenon was not limited to autoimmunity-prone NOD mice. In the transplanted mice, tetramer binding sites were examined and sites increased fivefold in NOD mice that had been transplanted with B6 skin. These results demonstrate that directly alloreactive T cells recognize an allogeneic MHC II molecule and a graft leukocyte peptide in the same way that microbe-reactive T cells recognize a syngeneic MHC II molecule and a microbial peptide. The data suggest that the potency of direct alloreactivity is not due to a high frequency of T cells that recognize MHC molecules alone, but rather the consequence of an additional weak activation of T cells, specific for hundreds of different graft leukocyte peptide-allogeneic MHC complexes. These data have important conceptual relevance in understanding basic mechanisms that drive alloimmunity and inflammation.

Modeling the Effect of the Aryl Hydrocarbon Receptor on Transplant Immunity.

BackgroundExposure to pollutants through inhalation is a risk factor for lung diseases including cancer, asthma, and lung transplant rejection, but knowledge of the effects of inhaled pollutants on pathologies outside of the lung is limited.MethodsUsing the minor-mismatched model of male C57BL/6J (B6) to female B6 skin grafts, recipient mice were treated with an inhaled urban dust particle sample every 3 days before and after grafting. Graft survival time was determined, and analysis of the resulting immune response was performed at time before rejection.ResultsSignificant prolongation of male skin grafts occurred in recipient female mice treated with urban dust particles compared with controls and was found to be dependent on aryl hydrocarbon receptor (AHR) expression in the recipient mouse. T cell responses to the male histocompatibility antigen (H-Y) Dby were not altered by exposure to pollutants. A reduction in the frequency of IFNγ-producing CD4 T cells infiltrating the graft on day 7 posttransplant was observed. Flow cytometry analysis revealed that AHR expression is upregulated in IFNγ-producing CD4 T cells during immune responses in vitro and in vivo.ConclusionsSurprisingly, inhalation of a pollutant standard was found to prolong graft survival in a minor-mismatched skin graft model in an AHR-dependent manner. One possible mechanism may be an effect on IFNγ-producing CD4 T cells responding to donor antigen. The increased expression of AHR in this CD4 T cell subset suggests that AHR ligands within the particulate matter may be directly affecting the type 1 T helper cell response in this model.

Inhibition of JAK/STAT Signaling Synergizes With CTLA4-Ig to Promote Transplant Tolerance.

PURPOSE: Transplant tolerance induction through conventional costimulation blockade (CoB) remains an elusive goal. Recent evidence suggests that inflammatory cytokines (IC) contribute to the activation of alloreactive T cells in a CD28- and CD40-independent manner. We aimed to understand the mechanisms underlying this process and to delineate the possible synergism between CD28 blockade and inhibition of IC signaling via JAK/STAT inhibition. METHODS: Murine T cell activation was assessed by quantification of IL-2 secretion (at 24h post stimulation) and through a CFSE proliferation assay. Stimulation in an inflamed environment was simulated through addition of supernatant (MATSup) from maturing dendritic cells (following exposure to LPS). Jak inhibition was exerted with Tofacitinib (Tofa). Differential expression of DC maturation markers in response to LPS +/- Tofa was analyzed by flow cytometry. In vivo synergism between Tofa and CTLA4-Ig was tested in a BALB/c to B6 skin graft model. RESULTS: Activation-associated early IL-2 production by T cells was diminished by CTLA4-Ig. The presence of MATSup, however, completely abolished this effect. Similarly, MATSup counteracted the anti-proliferative effect of CTLA4-Ig on both CD4 and CD8 T cells in our CFSE-based assay. Tofa addition was able to completely restore CTLA4-Ig inhibition in presence of MATSup. Tofa also reduced DC expression of costimulatory molecules (CD40, CD80, and CD86) in response to LPS, while preserving high MHC II expression. Our skin transplant model showed that a short course of Tofa (d0-12) synergized with CTLA4-Ig doubling skin allograft survival (MST untreated: 9.5d, CTLA4-Ig only: 13d, Tofa+CTLA4: 18d). CONCLUSION: Our results clearly indicate that the stimulatory effect of inflammatory cytokines counteracts the efficacy of CoB. However, their redundant activity can be neutralized through broad inhibition of Jak signaling that targets both T cells and DC. Our transplant survival data suggest that combining Jak inhibition with CoB is a promising strategy for promotion of transplant acceptance.

CD27low NK Cells Prolong Allograft Survival By Inhibiting Alloreactive CD8+ T Cell Responses.

While NK cells are emerging as key effector cells in alloimmune responses, the underlying mechanisms by which distinct NK cell subsets promote either allograft rejection or tolerance remain unclear. Therefore, the purpose of this study was to investigate the role of distinctively mature NK cell subsets in alloimmunity in the context of their regulation by T-box-transcription factors. Since T-bet controls the maturation of NK cells from immature CD27high to terminally-differentiated CD27low NK cells, we created BALB/c Rag-KO mice on a T-bet-KO background to study the different roles of CD27low vs CD27high NK cells in a model of T cell-mediated allograft rejection. For this, Rag-T-bet double-KO (DKO) and Rag-KO recipients of fully mismatched B6 skin allografts (STx) were adoptively transferred with purified T cells from Foxp3-gfp-knockin BALB/c mice. Groups of recipient mice were treated with CTLA4Ig and anti-CD154 (MR-1) on day 0, 2, 4 and 6 after reconstitution. Alloimmune responses were analyzed by histology, ELISA, RT-PCR and flow cytometry. First, we found by flow cytometry that Rag-T-bet DKO, but not Rag-KO, mice fully lack mature CD27lowNKp46+ NK cells in the periphery, confirming that our model allows us to study different NK cell subsets in alloresponses. Importantly, T cell-reconstituted Rag-KO recipients (with CD27low NK cells) showed significantly prolonged allograft survival upon costimulatory blockade (MST 35±3.4d) when compared to DKO mice lacking CD27low NK cells (28±1.2d), indicating that CD27low NK cells promote allograft survival in Rag-KO mice. Critically, in the DKO recipients memory CD8+ T cells showed a strikingly increased proliferation response (CFSE assay) and higher IFN-γ production (3.7±0.5 vs 1.7±0.5 × 106 IFN-γ+ cells). Therefore, we hypothesized that CD27low NK cells regulate alloreactive CD8+ T cell responses under costimulatory blockade conditions. To test this, we transferred CD27low NK cells into DKO STx recipients and found that reconstitution with CD27low NK cells restores prolonged allograft survival (40.4±5.0d vs 29.6±0.6d) and reduces the proliferation of alloreactive IFN-γ+CD8+ T cells. We conclude that mature CD27low NK cells promote allograft survival under costimulatory blockade conditions by inhibiting alloreactive CD8+ T cell responses.

Inhibition of Jak/STAT signaling synergizes with CTLA4-Ig to promote transplant acceptance (IRC2P.454)

Abstract Transplant tolerance induction through conventional costimulation blockade (CoB) remains an elusive goal. Recent evidence suggests that inflammatory cytokines (IC) contribute to the activation of alloreactive T cells in a CD28- and CD40-independent manner. We aimed to understand the mechanisms underlying this process and delineating the possible synergism between CD28 blockade and inhibition of IC signaling via Jak/STAT inhibition. Murine T cell activation was assessed by quantification of IL-2 secretion (at 24h post TCR/CD28 engagement). CTLA4-Ig diminished the proportion of IL-2+ cells. However, the concomitant addition of the supernatant (MATSup) from maturing dendritic cells completely abolished this effect. This suggested an early impact of IC on T cell activation. In a CFSE-based proliferation assay, MATSup counteracted the anti-proliferative effect of CTLA4-Ig on both CD4 and CD8 T cells. Inhibition of Jak/STAT signaling through addition of Tofacitinib (Tofa) restored CTLA4-Ig regulation in presence of MATSup. Finally, in a BALB/c to B6 skin transplant model a short course of Tofa (d0-12) synergized with CTLA4-Ig doubling skin allograft survival (MST untreated: 9.5d, CTLA4-Ig only: 13d, Tofa+CTLA4: 18d). Overall, our results indicate that the stimulatory effect of IC counteracts the efficacy of CoB. However, their redundant activity can be neutralized through concomitant inhibition of Jak signaling - a promising strategy for promotion of transplant acceptance.

Interleukin 5 immunotherapy depletes alloreactive plasma cells

BackgroundLong-lived plasma cells (PCs) that form after alloantigen sensitization produce donor-specific alloantibodies that generate a positive serum crossmatch and preclude transplantation. New approaches for desensitization, including PC depletion with proteasome inhibition, show promise but carry considerable toxicity. Recently, eosinophils have been shown to govern PC persistence. Interleukin 5 (IL-5) depletion is known to reduce eosinophils in human asthmatics. We hypothesized that treatment with an anti-IL-5 antibody can deplete alloreactive PCs, reduce donor-specific alloantibodies, and serve as a less toxic alternative to proteasome inhibition. MethodsBALB/c mice were sensitized with B6 skin allografts. Starting at 8 wk after sensitization, control mice received injections of phosphate-buffered saline, whereas experimental mice received weekly injections of an anti-IL-5 antibody. PCs were enumerated by enzyme-linked immunosorbent spot after 8 wk. ResultsAll control and experimental recipients of skin allografts developed positive crossmatches when screened at 8 wk after sensitization. All experimental mice treated with anti-IL-5 showed a reduction in their total PC numbers. Also, in contrast to the known adverse effects of proteasome inhibition, experimental mice treated with anti-IL-5 exhibited negligible weight loss or lymphopenia. ConclusionsTreatment with anti-IL-5 is sufficient to reduce, but not eliminate, alloreactive PCs in the bone marrow. This is because of the targeted reduction of eosinophils leading to a reduction in the PC survival factors a proliferation-inducing ligand and IL-6. Generalized toxicity was not observed in experimental mice. Overall, IL-5 directed immunotherapy can eliminate PC's but is unlikely to be a clinically significant desensitization strategy given the persistence of DSA

CD4 + Regulatory T Cells Generated in Vitro with IFN-γ and Allogeneic APC Inhibit Transplant Arteriosclerosis

We have developed a method to generate alloreactive regulatory T cells in vitro in the presence of interferon (IFN)-gamma and donor antigen presenting cells (APCs). We hypothesized that these IFN-gamma-conditioned T cells (Tcon) would reduce transplantation-associated arteriosclerosis. Tcon were generated from mouse (CBA.Ca, H-2(k)) CD4(+) T cells cultured in the presence of IFN-gamma for 14 days. These cultures were pulsed with bone marrow-derived B6 (H-2(b)) APC. 1 x 10(5) CD25(-)CD4(+) effector T cells from naive H-2(k) mice were then cotransferred with 4 x 10(5) Tcon into CBA-rag(-/-) mice. One day later, these mice received a fully allogenic B6 CD31(-/-) abdominal aorta transplant. Transfer of CD25(-)CD4(+) effectors resulted in 29.7 +/- 14.5% luminal occlusion of allogeneic aortic grafts after 30 days. Cotransfer of Tcon reduced this occlusion to 11.7 +/- 13.1%; P < 0.05. In addition, the CD31(-) donor endothelium was fully repopulated by CD31(+) recipient endothelial cells in the absence of Tcon, but not in the presence of Tcon. In some experiments, we cotransplanted B6 skin with aortic grafts to ensure enhanced reactivation of the regulatory cells, which led to an additional reduction in vasculopathy (1.9 +/- 3.0% luminal occlusion). In the presence of Tcon, CD4(+) T cell infiltration into grafts was markedly reduced by a regulatory mechanism that included reduced priming and proliferation of CD25(-)CD4(+) effectors. These data illustrate the potential of ex vivo generated regulatory T cells for the inhibition of transplant-associated vasculopathy.

The myth of the subclinical rejection: Is it real

Introduction: Subclinical rejection (SCAR) of renalallografts refers to graft lymphocytic infiltrationtaking acute rejection histologic pattern despitestable renal function. There are no data to suggestthat subclinical tubulointerstitial inflammation isregulatory or in any way beneficial to the graft. Wehave investigated whether C57BL/6 CD8 T cellshome to long term engrafted (LTE) DBA/2 skinallografts and if it is protecting or rejecting. Methods and results: We transplanted two groupsof B6 CD4 KO mice, 6 mice each, with MHCmismatched DBA/2 skin. Only the 1st group wastreated with Rapamycin (RPM) as reported. After100 days of LTE, we challenged RPM treated hostswith a 2nd DBA/2 skin graft. The 2nd but not the 1stgraft was rejected. Then we investigated thefunctional effects of graft inflitrating CD8 T cells.DBA/2 skin grafts were harvested 100 daysposttransplantation from (i) RPM treated B6 CD4ko mice (N=5) and (ii) skin autografts (N=5) inDBA/2 recipients. LTE DBA/2 allografts or controlDBA/2 autografts were then transplanted ontoC57BL/6-Rag KO hosts, and peripheral bloodlymphocytes (PBL) samples were collected 30 dayspost skin transplantation. CD8T cells can not bedetected in PBL of nae RAG-/- mice. 4.6 % CD8T cells are detected in PBL of RAG-/- recipients ofLTE allografts, but not in recipients of syngeneicgrafts. To test the protective function of the grafthoming CD8 T cells (from LTE RPM mice) thatexpanded by homeostatic proliferation and arepresent in PBL of the RAG-/-, 0.2 x 106 CD8 Tcells from naive CD4KO mice were adoptivelytransferred into the RAG-/- hosts bearing the LTEDBA/2 allografts or DBA/2 autografts 30 daysfollowing skin transplantation. Survival of LTEDBA/2 skin allografts transplanted onto RAG-/-mice were significantly prolonged. Conclusion: Graft infiltrating CD8 T cells areregulatory and functionally active to protectallograft from rejection.

Costimulatory Blockade Enhances Engraftment and Prevents Anti-Donor Antibody Generation in Nonmyeloablative Conditioned Prediabetic NOD Mice

Chimerism induces tolerance to organ and tissue grafts. Although the toxicity of conditioning has been significantly reduced with nonmyeloablative approaches, it would be beneficial to further reduce the intensity of conditioning in organ transplant recipients. Studies in normal mice and autoimmune NOD mice have revealed that the primary role for conditioning is to control host-versus-graft alloreactivity, allowing for immune-based conditioning to establish chimerism. Recent insights into the mechanism of interaction and crossregulation between innate and adaptive immune responses have led to the development of new approaches for immune-based conditioning. We previously demonstrated that preconditioning of diabetes-prone NOD mice with anti-CD8 monoclonal antibody (mAb) combined with anti-CD154 mAb allowed chimerism to be established with 550 cGy total body irradiation (TBI). Here we investigated whether combined costimulatory blockade can improve the nonmyeloablative regimen and enhance bone marrow chimerism in autoimmune recipients. Prediabetic NOD (H-2d) mice were treated with anti-CD8 antibody (day -3). After 500 cGy TBI (day 0), recipient NOD mice were transplanted with 30 × 106 B10.BR (H-2k) bone marrow cells (day 0) and received anti-CD154, anti-OX40L, and anti- inducible co-stimulatory molecule (ICOS) mAbs intraperitoneally (IP) (day 0, 1, 2, 3). Chimerism and multilineage analysis were quantitated by flow cytometry. Chimeric animals produced multilineage donor chimerism. While 18 prediabetic NOD mice demonstrated an average PB chimerism of ~37% at 1 month and 61% engraftment, 15 of 18 mice lost PB chimerism by 6 months. NOD mice were then transplanted with B10.BR donor skin grafts 1 month post BM transplant. Flow cytometry cross-match assays were performed to detect anti-donor antibody (Ab) in the sera collected 1 month post skin grafting. 44% of chimeric NOD mice (n=9) accepted their skin grafts for over 90 days, while 100% of non-chimeric animals (n=4) rejected their grafts within 60 days. NOD mice transplanted with B10.BR skin grafts alone rejected their grafts within 2 weeks and generated significantly higher levels of donor-specific Abs (mean fluorescence intensity: 251.2 ± 61.5, P<0.0001, n=6) compared to chimeric NOD (4.5 ± 0.6, P<0.0005, n=17) or non-chimeric NOD (12.6 ± 2.4, P<0.0005, n=10). Both chimeric NOD and non-chimeric NOD mice transplanted with 3rd party B6 skin grafts rejected their grafts within 30 days. These results suggest that rejection of skin grafts in chimeric and nonchimeric animals is not associated with anti-donor antibody generation in preconditioned animals. Studies are currently underway to determine if impaired generation of effector/memory T cells (CD44high/CD62Llow/−) is responsible for enhanced engraftment and to determine the potential mechanisms that promote tolerance in prediabetic NOD mice after loss of PB chimerism.

Induction of regulatory T cells from mature T cells by allogeneic thymic epithelial cells in vitro

The ability of thymic epithelial cells (TEC) to re-educate mature T cells to be regulatory T cells has not been addressed. In the present study, this issue was directly investigated by co-culturing of mature T cells and allo-TECs. B6 macrophage cell line 1C21-cultured BALB/c splenocytes responded to B6 antigens in vitro. However, BALB/c splenocytes precultured with B6-derived TECs 1-4C18 or 1C6 did not proliferate to B6 antigens, but responded to rat antigens. Exogenous interleukin-2 (IL-2) failed to revise the unresponsiveness of these T cells. Allo-TEC-cultured T cells predominantly expressed Th2 cytokines (IL-4 and IL-10). B6 TEC-cultured BALB/c splenocytes markedly inhibited the immune responses of naïve BALB/c splenocytes to B6 antigens, but not to rat or the third-party mouse antigens. BALB/c nude mice that received naïve syngeneic splenocytes rejected B6 or rat skin grafts by 17 days postskin grafting; however, co-injection of B6 TEC-cultured BALB/c splenocytes significantly delayed B6 skin graft rejection (P < 0.01), with the unchanged rejection of rat skin grafts. These studies demonstrate that allo-TECs are able to 'educate' mature T cells to be regulatory cells, and suggest that regulatory cells derived from mature T cells by TECs may play an important role in T cell tolerance to allo- and auto-antigens.

Frequency of Natural Regulatory CD4+CD25+ T Lymphocytes Determines the Outcome of Tolerance across Fully Mismatched MHC Barrier through Linked Recognition of Self and Allogeneic Stimuli

We show in this study that long-term tolerance to allogeneic skin grafts can be established in the absence of immunosuppression by the combination of the following elements: 1) augmenting the frequency of regulatory CD4(+)CD25(+) T cells (Treg) and 2) presentation of the allogeneic stimuli through linked recognition of allo- and self-epitopes on semiallogeneic F(1) APCs. BALB/c spleen cells enriched for CD4(+)CD25(+) T lymphocytes were transferred either to BALB/c nu/nu mice or to BALB/c nu/nu previously injected with F(1)(BALB/c x B6.Ba) spleen cells, or else grafted with F(1)(BALB/c x B6.Ba) skin (chimeric BALB/c nu/nu-F(1)). Chimeric BALB/c nu/nu-F(1) reconstituted with syngeneic CD25(+)-enriched spleen cells were unable to reject the previously transferred F(1)(BALB/c x B6.Ba) spleen cells or F(1)(BALB/c x B6.Ba) skin grafts, and a specific tolerance to a secondary B6 graft was obtained, with rejection of third-party CBA grafts. BALB/c nu/nu mice reconstituted only with syngeneic CD25(+)-enriched spleen cells rejected both B6 and CBA skin grafts. In contrast, when chimeric BALB/c nu/nu-F(1) were reconstituted with spleen populations comprising normal frequencies of Treg cells, the linked recognition of allo and self resulted in breaking of self tolerance and rejection of syngeneic grafts, strongly suggesting that linked recognition works in both directions, either to establish tolerance to allo, or to break tolerance to self, the critical parameter being the relative number of Treg cells.

Acceptance of related and unrelated cardiac allografts in neonatally tolerized mice is cardio-specific and transferable by regulatory CD4 + T cells

Non-donor-specific cardiac allograft acceptance is induced in C3H/He (C3H; H-2k) recipients injected as neonates with allogeneic BALB/c (BALB; H-2d) fetal liver cells (FLC). This occurs despite intact reactivity to donor-type and third-party alloantigens in in vitro assays and skin transplants. To investigate a role for regulatory T cells, we performed adoptive transfer studies and specifically assessed CD4+ and CD4- T cells. Three cell populations (splenocytes, CD4+, CD4-) derived from neonatally-treated mice with accepted C57BL/6 (B6; H-2b) third-party cardiac grafts were adoptively transferred into sub-lethally-irradiated C3H mice. Reconstituted mice were challenged with B6 cardiac grafts, B6 skin grafts, or unrelated cardiac grafts. Separated cells were assessed in vitro. B6, BALB, and NZW (H-2z) graft acceptance was transferred by unfractionated splenocytes. CD4+ cells transferred B6 graft acceptance (85% survival > 100 days). CD4- cells, unfractionated cells from naive or only irradiated mice, and unfractionated cells from neonatally-treated non-transplanted C3H mice rejected grafts within 35 days. No inoculum induced skin graft acceptance. Co-cultured assays confirmed the suppressive function of CD4+ cells in vitro. Cardiac allograft acceptance in our model is regulated by CD4+ cells. The regulatory cell population is induced by the cardiac graft itself and mediates in vivo cardiac graft acceptance in a tissue-specific but not donor-strain-specific manner.

Chimeric donor cells play an active role in both induction and maintenance phases of transplantation tolerance induced by mixed chimerism.

Donor hemopoietic cell engraftment is considered to be an indicator of allograft tolerance. We depleted chimerism with cells specifically presensitized to the bone marrow donor to investigate its role in mixed chimera-induced tolerance. Three experimental models were used: model A, B10.A cells presensitized to B6 (a anti-b cells) were injected into (B6 x D2)F(1) --> B10.A mixed chimeras grafted with DBA/2 skin; model B, anti-B6 presensitized cells prepared in DBA/2 --> B10.A mixed chimeras, thus unresponsive to DBA/2 (a anti-b/tol-d cells), were injected into (B6 x D2)F(1) --> B10.A mixed chimeras grafted with DBA/2 skin; and model C, (BALB/c x B6)F(1) cells presensitized to CBA (d/b anti-k cells) were injected into (B6 x CBA)F(1) --> BALB/c mixed chimeras grafted with B6 skin. Skin was grafted on day 30. Injection of each cell type before skin grafting abolished hemopoietic cell engraftment and prevented allograft acceptance. Injection of presensitized cells after skin grafting resulted in different outcomes depending on the models. In model A, injection of a anti-b cells completely depleted chimerism and caused allograft rejection. In model B, injection of a anti-b/tol-d cells markedly reduced, but did not deplete, peripheral chimerism and maintained skin allograft survival. In model C, d/b anti-k cells reduced chimerism to the background levels but failed to cause graft rejection, probably due to persistence of injected cells which share MHC with skin grafts. Together, the results show that presence of chimeric donor cells is essential in both the induction and maintenance phases of tolerance induced by mixed chimerism.

Immune Tolerance in Murine Islet Transplantation Across HY Disparity

Background: Minor histocompatibility HY antigen, as a transplantation antigen, has been known to cause graft rejection in MHC (major histocompatibility complex) matched donor-recipient. The aim of our study is to investigate the role of male antigen (HY) disparity on MHC matched pancreatic islet transplantation and to examine the mechanism of the immune reaction. Methods: Pancreatic islets were isolated and purified by collagen digestion followed by Ficoll gradient. The isolated islets of male C57BL6/J were transplanted underneath the kidney capsule of syngeneic female mice rendered diabetic with streptozotocine. Blood glucose was monitored for the rejection of engrafted islets. After certain period of time, tail to flank skin transplantation was performed either on mouse transplanted with HY mismatched islets or on sham treated mouse. The rejection was monitored by scoring gross pathology of the engrafted skin. Results: HY mismatched islets survived more than 300 days in 14 out of 15 mice. The acceptance of second party graft (male B6 islets) and the rejection of third party graft (male BALB/c islets) in these mice suggested the tolerance to islets with HY disparity. B6 Skin with HY disparity was rejected on day . However, HY mismatched skin transplanted on the mice tolerated to HY mismatched islets survived more than 240 days. Tetramer staining in these mice indicated the CTL recognizing MHC Db/Uty was not deleted or anergized. Conclusion: The islet transplantation across HY disparity induced tolerance to HY antigen in C57BL6 mouse, which in turn induced tolerance to HY mismatched skin, which otherwise would be rejected within 25 days. The MHC tetramer staining suggested the underlying mechanisms would not be clonal deletion or anergy.

Nonmyeloablative conditioning is sufficient to allow engraftment of EGFP-expressing bone marrow and subsequent acceptance of EGFP-transgenic skin grafts in mice

AbstractImmunologic reactions against gene therapy products may prove to be a frequent problem in clinical gene therapy protocols. Enhanced green fluorescence protein (EGFP) is commonly used as a marker in gene transfer protocols, and immune responses against EGFP-expressing cells have been documented. The present study was designed to investigate the effect of a pharmacologic, nonmyeloablative, conditioning regimen on the development of EGFP+ donor/recipient mixed bone marrow chimerism and ensuing tolerance to EGFP-expressing transplants. To this end, C57BL/6J (B6) mice were treated with soluble formulations of either busulfan (Busulfex) or the closely related compound treosulfan, followed by transplantation of bone marrow cells from EGFP-transgenic (B6-EGFP.Tg) donor mice. Such conditioning regimens resulted in long-term persistence of donor EGFP+ cells among various hematopoietic lineages from blood, bone marrow, and thymus. Stable hematopoietic chimeras transplanted at 10 to 17 weeks after bone marrow transplantation (BMT) with B6-EGFP.Tg skin grafts all accepted their transplants, whereas non-EGFP chimeric B6 control animals were able to mount rejection of the EGFP+ B6 skin grafts. Control third-party grafts from major histocompatibility complex (MHC)–mismatched mice were rejected within 20 days, indicating that acceptance of EGFP-expressing skin grafts was the result of specific immune tolerance induction by the transplantation of EGFP-transgenic bone marrow. Long-term tolerance to EGFP in chimeric recipients was confirmed by the absence of anti-EGFP–reactive T cells and antibodies. These results broaden the therapeutic potential for using hematopoietic molecular chimerism in nonmyeloablated recipients as a means of preventing rejection of genetically modified cells.

Prolongation of allogeneic skin graft survival by injection of anti-Ly49A monoclonal antibody YE1/48

Ly49A receptors expressed on NK, NKT, and T cells play inhibitory roles in regulating the immune responses in vivo and in vitro. Whether or not injection of anti-Ly49A monoclonal antibody (mAb) YE1/48 can block allograft rejection has not been evaluated. Balb/c mouse recipients received intraperitoneal injections of YE1/48 mAb (0.5 mg) or control mAb or phosphate-buffered saline on days −1 and 10. On day 0, fully MHC-mismatched allogeneic C57BL/6 (B6) skin grafts were implanted. The skin graft survival and anti-donor humoral responses were observed. Whereas allogeneic B6 skin grafts survived 14 days in isotopy control antibody-treated or nontreated Balb/c mice, injection of YE1/48 mAb significantly prolonged B6 skin graft survival to 19 days ( P < 0.0005). Injection of YE1/48 mAb into presensitized Balb/c recipients did not significantly delay B6 skin graft rejection. On the other hand, after depleting recipient NK, NKT, and some cytotoxic T cells by injection of anti-asialo GM1, YE1/48 failed to prolong B6 skin graft survival in Balb/c recipients. The present studies indicate that injection of YE1/48 mAb significantly delays allogeneic skin graft rejection in nonsensitized recipients but not in sensitized recipients. The presence of NK, NKT cells, and some cytotoxic T cells may be essential for YE1/48-mediated immunosuppression in vivo.

Ly-6A is critical for the function of double negative regulatory T cells.

We have recently demonstrated that CD3+CD4-CD8- double negative (DN) T cells can down-regulate allogeneic immune responses both in vitro and in vivo by killing activated syngeneic CD8+ T cells. The goal of this study was to identify molecules that are crucial for DN T cell-mediated suppression. We demonstrate that Ly-6A (Sca-1) is highly expressed on DN T cells. Incubation with IL-10 significantly reduced Ly-6A expression and the function of DN T cells. DN T cell-mediated killing was significantly reduced when Ly-6A was blocked.Ly-6A-deficient mice showed an accelerated allograft rejection when compared to wild-type controls. Furthermore we demonstrate that pretransplantation donor lymphocyte infusion (DLI) led to activation and proliferation of recipient DN T cells and prolongation of bm1-->B6 skin allograft survival. However, when the recipients were deficient in Ly-6A, the beneficial effect of DLI on allograft survival was abolished. Moreover, deficiency in Ly-6A did not affect the activation and proliferation of DN T cells. Rather, it impaired the ability of DN T cells to kill activated anti-donor CD8+ T cells. Taken together, our data indicate that Ly-6A plays a crucial role in DN T cell-mediated regulation in vitro and in vivo, perhaps by enhancing DN-CD8+ T cell signaling.

Development of autoimmunity after skin graft rejection via an indirect alloresponse.

T cell allorecognition occurs through direct contact with donor peptide: MHC complexes on graft cells and through indirect recognition of donor-derived determinants expressed by recipient MHC molecules. As both indirect allorecognition and autoantigen recognition are self-restricted, we hypothesized that chronic activation of indirectly primed T cells might result in determinant spreading to involve autoantigens, analogous to that which occurs during chronic autoimmune diseases. We placed C57BL/6 MHC II knockout (B6 II-/-) skin grafts onto BALB/c SCID mice reconstituted with wild-type (WT) CD4+ T cells. Under these conditions the CD4+ cells could not recognize any antigen on the graft, but could respond through the indirect pathway. CD4+ cell-mediated rejection of WT B6 skin was studied to determine if autoreactivity was induced after direct allorecognition. Recall immune responses against donor- and self-stimulator cells were determined by ELISPOT and animals were tested for their ability to reject second isografts. WT allografts were rejected by day 14 although B6 II-/- grafts underwent delayed rejection over 4-5 weeks. CD4+ cells reisolated from the recipients of the MHC II-/- grafts, but not from the recipients of WT grafts, vigorously produced interferon-gamma and interleukin-2 in response to self, BALB/c stimulators. These autoreactive CD4+ T cells mediated rejection of a second isogenic BALB/c skin graft, demonstrating that the autoimmune response was pathogenic. Autoreactivity can develop after transplant rejection via the indirect pathway. Although the direct alloresponse is likely to be the driving force in acute graft rejection, posttransplantation induced autoimmune responses may be important elements of delayed or chronic rejection.