CD154 Blockade Abrogates Allospecific Responses and Enhances CD4+ Regulatory T‐Cells in Mouse Orthotopic Lung Transplant

Acute cellular rejection (ACR) is a common and important clinical complication following lung transplantation. While there is a clinical need for the development of novel therapies to prevent ACR, the regulation of allospecific effector T‐cells in this process remains incompletely understood. Using the MHC‐mismatched mouse orthotopic lung transplant model, we investigated the short‐term role of anti‐CD154 mAb therapy alone on allograft pathology and alloimmune T‐cell effector responses. Untreated C57BL/6 recipients of BALB/c left lung allografts had high‐grade rejection and diminished CD4⁺: CD8⁺ graft ratios, marked by predominantly CD8⁺>CD4⁺ IFN‐γ⁺ allospecific effector responses at day 10, compared to isograft controls. Anti‐CD154 mAb therapy strikingly abrogated both CD8⁺ and CD4⁺ alloeffector responses and significantly increased lung allograft CD4⁺: CD8⁺ ratios. Examination of graft CD4⁺ T‐cells revealed significantly increased frequencies of CD4⁺CD25⁺Foxp3⁺ regulatory T‐cells in the lung allografts of anti‐CD154‐treated mice and was associated with significant attenuation of ACR compared to untreated controls. Together, these data show that CD154/CD40 costimulation blockade alone is sufficient to abrogate allospecific effector T‐cell responses and significantly shifts the lung allograft toward an environment predominated by CD4⁺ T regulatory cells in association with an attenuation of ACR.     

Sirolimus Enhances the Magnitude and Quality of Viral‐Specific CD8+ T‐Cell Responses to Vaccinia Virus Vaccination in Rhesus Macaques

Sirolimus is a potent antiproliferative agent used clinically to prevent renal allograft rejection. However, little is known about the effects of maintenance immunosuppressive agents on the immune response to potentially protective vaccines. Here we show that sirolimus paradoxically increases the magnitude and quality of the CD8⁺ T‐cell response to vaccinia vaccination in nonhuman primates, fostering more robust recall responses compared to untreated and tacrolimus‐treated controls. Enhancement of both the central and effector memory compartments of the vaccinia‐specific CD8⁺ T‐cell response was observed. These data elucidate new mechanistic characteristics of sirolimus and suggest immune applications extending beyond its role as an immunosuppressant.     

Recipient Dendritic Cells,But Not B Cells,Are Required Antigen‐Presenting Cells for Peripheral Alloreactive CD8+ T‐Cell Tolerance

Induction of mixed allogeneic chimerism is a promising approach for achieving donor‐specific tolerance, thereby obviating the need for life‐long immunosuppression for solid organ allograft acceptance. In mice receiving a low dose (3Gy) of total body irradiation, allogeneic bone marrow transplantation combined with anti‐CD154 tolerizes peripheral CD4 and CD8 T cells, allowing achievement of mixed chimerism with specific tolerance to donor. With this approach, peripheral CD8 T‐cell tolerance requires recipient MHC class II, CD4 T cells, B cells and DCs. Recipient‐type B cells from chimeras that were tolerant to donor still promoted CD8 T‐cell tolerance, but their role could not be replaced by donor‐type B cells. Using recipients whose B cells or DCs specifically lack MHC class I and/or class II or lack CD80 and CD86, we demonstrate that dendritic cells (DCs) must express CD80/86 and either MHC class I or class II to promote CD8 tolerance. In contrast, B cells, though required, did not need to express MHC class I or class II or CD80/86 to promote CD8 tolerance. Moreover, recipient IDO and IL‐10 were not required. Thus, antigen presentation by recipient DCs and not by B cells is critical for peripheral alloreactive CD8 T cell tolerance.     

CD8+ T‐Cell Depletion and Rapamycin Synergize with Combined Coreceptor/Stimulation Blockade to Induce Robust Limb Allograft Tolerance in Mice

The growing development of composite tissue allografts (CTA) highlights the need for tolerance induction protocols. Herein, we developed a mouse model of heterotopic limb allograft in a stringent strain combination in which potentially tolerogenic strategies were tested taking advantage of donor stem cells in the grafted limb. BALB/c allografts were transplanted into C57BL/6 mice treated with anti‐CD154 mAb, nondepleting anti‐CD4 combined to either depleting or nondepleting anti‐CD8 mAbs. Some groups received additional rapamycin. Both depleting and nondepleting mAb combinations without rapamycin only delayed limb allograft rejection, whereas the addition of rapamycin induced long‐term allograft survival in both combinations. Nevertheless, robust donor‐specific tolerance, defined by the acceptance of a fresh donor‐type skin allograft and simultaneous rejection of third‐party grafts, required initial CD8⁺ T‐cell depletion. Mixed donor‐recipient chimerism was observed in lymphoid organs and recipient bone marrow of tolerant but not rejecting animals. Tolerance specificity was confirmed by the inability to produce IL‐2, IFN‐γ and TNF‐α in MLC with donor antigen while significant alloreactivity persisted against third‐ party alloantigens. Collectively, these results show that robust CTA tolerance and mixed donor‐recipient chimerism can be achieved in response to the synergizing combination of rapamycin, transient CD8⁺ T‐cell depletion and costimulation/coreceptor blockade.     

Differential Requirement of CD27 Costimulatory Signaling for Naïve Versus Alloantigen‐Primed Effector/Memory CD8+ T Cells

CD8⁺ memory T cells endanger allograft survival by causing acute and chronic rejection and prevent tolerance induction. We explored the role of CD27:CD70 T‐cell costimulatory pathway in alloreactive CD8⁺/CD4⁺ T‐cell activation. CD27‐deficient (CD27^?/?) and wild‐type (WT) B6 mice rejected BALB/c cardiac allografts at similar tempo, with or without depletion of CD4⁺ or CD8⁺ T cells, suggesting that CD27 is not essential during primary T‐cell alloimmune responses. To dissect the role of CD27 in primed effector and memory alloreactive T cells, CD27^?/? or WT mice were challenged with BALB/c hearts either 10 or 40 days after sensitization with donor‐type skin grafts. Compared to WT controls, allograft survival was prolonged in day 40‐ but not day 10‐sensitized CD27^?/? recipients. Improved allograft survival was accompanied by diminished secondary responsiveness of memory CD8⁺ T cells, which resulted from deficiency in memory formation rather than their lack of secondary expansion. Chronic allograft vasculopathy and fibrosis were diminished in CD27^?/? recipients of class I‐ but not class II‐mismatched hearts as compared to WT controls. These data establish a novel role for CD27 as an important costimulatory molecule for alloreactive CD8⁺ memory T cells in acute and chronic allograft rejection.     

Inhibition of Innate Co‐Receptor TREM‐1 Signaling Reduces CD4+ T Cell Activation and Prolongs Cardiac Allograft Survival

The innate receptor “triggering‐receptor‐expressed‐on‐myeloid‐cells‐1” (TREM‐1) enhances downstream signaling of “pattern recognition receptor” (PRR) molecules implicated in inflammatory responses. However the mechanistic role of TREM‐1 in chronic heart rejection has yet to be elucidated. We examined the effect of TREM‐1⁺ antigen‐presenting cells (APC) on alloreactive CD4⁺ lymphocytes. Bm12 donor hearts were transplanted into wild‐type MHC‐class‐II‐mismatched C57BL/6J recipient mice. Progressive allograft rejection of bm12‐donor hearts with decreased organ function, severe vasculopathy and allograft fibrosis was evident within 4 weeks. TREM‐1⁺CD11b⁺MHC‐II⁺F4/80⁺CCR2⁺ APC and IFNγ‐producing CD4⁺ cells were detected during chronic rejection. Peptide inhibition of TREM‐1 attenuated graft vasculopathy, reduced graft‐infiltrating leukocytes and prolonged allograft survival, while being accompanied by sustained low levels of CD4⁺ and CD8⁺ cell infiltration. Remarkably, temporary inhibition of TREM‐1 during early immune activation was sufficient for long‐term allograft survival. Mechanistically, TREM‐1 inhibition leads to reduced differentiation and proliferation of IFNγ‐producing Th1 cells. In conclusion, TREM‐1 influences chronic heart rejection by regulating the infiltration and differentiation of CD4⁺ lymphocytes.     

Increased T Cell Glucose Uptake Reflects Acute Rejection in Lung Grafts

Although T cells are required for acute lung rejection, other graft–infiltrating cells such as neutrophils accumulate in allografts and are also high glucose utilizers. Positron emission tomography (PET) with the glucose probe [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) has been employed to image solid organ acute rejection, but the sources of glucose utilization remain undefined. Using a mouse model of orthotopic lung transplantation, we analyzed glucose probe uptake in the grafts of syngeneic and allogeneic recipients with or without immunosuppression treatment. Pulmonary microPET scans demonstrated significantly higher [¹⁸F]FDG uptake in rejecting allografts when compared to transplanted lungs of either immunosuppressed or syngeneic recipients. [¹⁸F]FDG uptake was also markedly attenuated following T cell depletion therapy in lung recipients with ongoing acute rejection. Flow cytometric analysis using the fluorescent deoxyglucose analog 2‐NBDG revealed that T cells, and in particular CD8⁺ T cells, were the largest glucose utilizers in acutely rejecting lung grafts followed by neutrophils and antigen‐presenting cells. These data indicate that imaging modalities tailored toward assessing T cell metabolism may be useful in identifying acute rejection in lung recipients.     

Proliferation of CD8‐Positive T Cells in Blood Vessels of Rat Renal Allografts

It is still disputed in which anatomical compartments of allograft recipients T‐cells proliferate. After experimental renal transplantation, host monocytes and lymphocytes accumulate in the lumina of graft blood vessels. In this study, we test the hypothesis that T lymphocytes proliferate in the vascular bed of the graft. Kidneys were transplanted in the Dark Agouti to Lewis rat strain combination, an established experimental model for acute rejection. Isogeneic transplantation was performed as a control. Cells in the S‐phase of mitosis were detected in situ three days posttransplantation by pulse‐labeling with BrdU and by immunohistochemical detection of the proliferating cell nuclear antigen (PCNA). More than 20% of all T‐cells in the lumina of allograft blood vessels incorporated BrdU and approximately 30% of them expressed PCNA. In the blood vessels of isografts as well as in other organs of allograft recipients, only few BrdU⁺ cells were detected. A majority of the BrdU⁺ cells in graft blood vessels expressed CD8. In conclusion, we demonstrate that CD8⁺ T lymphocytes proliferate in the lumina of the blood vessels of renal allografts during the onset of acute rejection.     

LFA‐1 Antagonism Inhibits Early Infiltration of Endogenous Memory CD8 T Cells into Cardiac Allografts and Donor‐Reactive T Cell Priming

Alloreactive memory T cells are present in virtually all transplant recipients due to prior sensitization or heterologous immunity and mediate injury undermining graft outcome. In mouse models, endogenous memory CD8 T cells infiltrate MHC‐mismatched cardiac allografts and produce IFN‐γ in response to donor class I MHC within 24 h posttransplant. The current studies analyzed the efficacy of anti‐LFA‐1 mAb to inhibit early CD8 T cell cardiac allograft infiltration and activation. Anti‐LFA‐1 mAb given to C57BL/6 6 (H‐2^b) recipients of A/J (H‐2^a) heart grafts on days –1 and 0 completely inhibited CD8 T cell allograft infiltration, markedly decreased neutrophil infiltration and significantly reduced intragraft expression levels of IFN‐γ‐induced genes. Donor‐specific T cells producing IFN‐γ were at low/undetectable numbers in spleens of anti‐LFA‐1 mAb treated recipients until day 21. These effects combined to promote substantial prolongation (from day 8 to 27) in allograft survival. Delaying anti‐LFA‐1 mAb treatment until days 3 and 4 posttransplant did not inhibit early memory CD8 T cell infiltration and proliferation within the allograft. These data indicate that peritransplant anti‐LFA‐1 mAb inhibits early donor‐reactive memory CD8 T cell allograft infiltration and inflammation suggesting an effective strategy to attenuate the negative effects of heterologous immunity in transplant recipients.     

The Jak Inhibitor CP‐690,550 Preserves the Function of CD4+CD25brightFoxP3+ Regulatory T Cells and Inhibits Effector T Cells

The Jak inhibitor CP‐690,550 inhibits alloreactivity and is currently being investigated for prevention of allograft rejection after transplantation. In this study, we examined the effect of CP‐690,550 on IL‐2‐mediated Jak/STAT5 phosphorylation by CD4⁺CD25^brightFoxP3⁺CD127^?/low T cells (Treg) and CD4⁺CD25^neg effector T cells (Teff) in kidney transplant (KTx) patients. Phosphospecific flow cytometry was used to study the effect of CP‐690,550 on IL‐2‐induced intracellular STAT5‐phosphorylation. IL‐2‐induced phosphorylation of STAT5 (P‐STAT5) in both Treg and Teff, which was significantly higher for CD4⁺CD25^bright Treg (increased by 71%, mean) than for CD4⁺CD25^neg Teff (increased by 42%). In the presence of 100 ng/mL CP‐690,550, a clinically relevant exposure, IL‐2‐induced P‐STAT5 was partially inhibited in CD4⁺CD25^brightTreg (% inhibition; 51%), while almost completely blocked in Teff (%inhibition; 84%, p = 0.03). The IC₅₀ was 2–3 times higher for Treg (104 ng/mL) than for Teff (40 ng/mL, p = 0.02). In the presence of CP‐690,550, Treg exhibited additional suppressive activities on the alloactivated proliferation of Teff (56%, mean). In addition, CD4⁺CD25^bright Treg from KTx‐patients receiving CP‐690,550 vigorously suppressed the proliferation of Teff (87%, mean). Our findings show that CP‐690,550 effectively inhibits Teff function but preserves the suppressive activity of CD4⁺CD25^bright regulatory T cells.     

Reversing Endogenous Alloreactive B Cell GC Responses With Anti‐CD154 or CTLA‐4Ig

Alloantibodies mediate acute antibody‐mediated rejection as well as chronic allograft rejection in clinical transplantation. To better understand the cellular dynamics driving antibody production, we focused on the activation and differentiation of alloreactive B cells in the draining lymph nodes and spleen following sensitization to allogeneic cells or hearts. We used a modified staining approach with a single MHC Class I tetramer (K^d) bound to two different fluorochromes to discriminate between the Class I‐binding and fluorochrome‐streptavidin‐binding B cells with a high degree of specificity and binding efficiency. By Day 7–8 postsensitization, there was a 1.5‐ to 3.2‐fold increase in the total numbers of K^d‐binding B cells. Within this K^d‐binding B cell population, approximately half were IgD^low, MHC Class II^high and CD86⁺, 30–45% expressed a germinal center (Fas⁺GL7⁺) phenotype and 3–12% were IRF4^hi plasma cells. Remarkably, blockade with anti‐CD40 or CTLA‐4Ig, starting on Day 7 postimmunization for 1 or 4 weeks, completely dissolved established GCs and halted further development of the alloantibody response. Thus MHC Class I tetramers can specifically track the in vivo fate of endogenous, Class I‐specific B cells and was used to demonstrate the ability of delayed treatment with anti‐CD154 or CTLA‐4Ig to halt established allo‐B cell responses.     

Selective Targeting of Human Alloresponsive CD8+ Effector Memory T Cells Based on CD2 Expression

Costimulation blockade (CoB), specifically CD28/B7 inhibition with belatacept, is an emerging clinical replacement for calcineurin inhibitor‐based immunosuppression in allotransplantation. However, there is accumulating evidence that belatacept incompletely controls alloreactive T cells that lose CD28 expression during terminal differentiation. We have recently shown that the CD2‐specific fusion protein alefacept controls costimulation blockade‐resistant allograft rejection in nonhuman primates. Here, we have investigated the relationship between human alloreactive T cells, costimulation blockade sensitivity and CD2 expression to determine whether these findings warrant potential clinical translation. Using polychromatic flow cytometry, we found that CD8⁺ effector memory T cells are distinctly high CD2 and low CD28 expressors. Alloresponsive CD8⁺CD2^hiCD28^? T cells contained the highest proportion of cells with polyfunctional cytokine (IFNγ, TNF and IL‐2) and cytotoxic effector molecule (CD107a and granzyme B) expression capability. Treatment with belatacept in vitro incompletely attenuated allospecific proliferation, but alefacept inhibited belatacept‐resistant proliferation. These results suggest that highly alloreactive effector T cells exert their late stage functions without reliance on ongoing CD28/B7 costimulation. Their high CD2 expression increases their susceptibility to alefacept. These studies combined with in vivo nonhuman primate data provide a rationale for translation of an immunosuppression regimen pairing alefacept and belatacept to human renal transplantation.     

Successful In Vitro Priming of EBV-Specific CD8+ T Cells Endowed with Strong Cytotoxic Function from T Cells of EBV-Seronegative Children

Epstein‐Barr virus (EBV)‐seronegative transplant recipients are at high risk of developing EBV‐associated post‐transplant lymphoproliferative disorder (PTLD), and would maximally benefit from an EBV‐directed T‐cell therapy for prevention or treatment of PTLD. So far, efforts to activate CD8+ EBV‐specific cytotoxic T lymphocytes (CTL) endowed with high specific cytotoxicity from EBV‐seronegative children have failed. We compared the CD8+ CTL priming efficiency of three different modified activation protocols, based on lymphoblastoid cell lines (LCL) stimulation potentially enhanced by either LCL presentation through dendritic cells, or selection of IFN‐γ+ cultured cells, or culture in the presence of rhIL‐12 and rhIL‐7, according to the standard protocol for reactivation of EBV‐specific CTL. We found that only specific LCL stimulation in the presence of rhIL‐12 and rhIL‐7 was able to reproducibly expand EBV‐specific CD8+ CTL endowed with strong cytotoxic activity from truly EBV‐seronegative children. The lines thus activated, which included specificities toward EBV latent and lytic proteins, showed high percentage CD8+ T cells, with <10% naïve CD8+/CCR7+/CD45RA+ cells. Overall, the total number of CD8+ central memory cells, and of CCR7 T‐cell effectors was comparable to that observed in healthy EBV‐seropositive controls. In conclusion, it is feasible to activate EBV‐specific CD8+ CTL with suitable characteristics for in vivo employment from EBV‐seronegative children.     

Measurement of CD4+ T‐cell function in predicting allograft rejection and recurrent hepatitis C after liver transplantation

Hashimoto K, Miller C, Hirose K, Diago T, Aucejo F, Quintini C, Eghtesad B, Corey R, Yerian L, Lopez R, Zein N, Fung J. Measurement of CD4+ T‐cell function in predicting allograft rejection and recurrent hepatitis C after liver transplantation.
Clin Transplant 2009 DOI: 10.1111/j.1399‐0012.2009.01169.x
© 2009 John Wiley & Sons A/S. Abstract: Recurrence of hepatitis C virus (HCV) can be difficult to distinguish from acute cellular rejection (ACR) following liver transplantation. The Cylex Immune Function Assay (ImmuKnow) provides objective measure of recipient’s immune function. The goal is to assess the ability of this assay to distinguish these similar conditions. A retrospective review was performed in 54 recipients with HCV. ImmuKnow assays were measured with allograft biopsies. Levels of adenosine triphosphate (ATP) release from CD4+ T cells (ng/mL) were compared with the following biopsy result classifications: 365 ± 130 with ACR (n = 11), 152 ± 100 with recurrent HCV (n = 26), 240 ± 71 with normal biopsies (n = 12), and 157 ± 130 with overlapping features of ACR and recurrent HCV (n = 5). Recipients with recurrent HCV had lower immune response than those with ACR (p < 0.0001).Using a cutoff level of 220, the sensitivity and specificity for distinguishing two conditions were 88.5% and 90.9%, respectively. When recipients with overlapping features had low immune response, three of four recipients’ subsequent biopsies showed recurrent HCV. In conclusion, the ImmuKnow assay can be a sensitive and specific additional test for distinguishing recurrent HCV from ACR and may be useful for predicting which recipients may be most vulnerable to recurrent HCV.     

CD4/CD8 T‐Cell Ratio in Peritoneal Dialysis Effluents Predicts the Outcome of Peritonitis in Patients Undergoing Continuous Ambulatory Peritoneal Dialysis

This study aimed to clarify the role of peritoneal T‐lymphocytes in peritoneal immune defense mechanisms. This study was designed to examine the changes in T‐cell subpopulations during peritonitis in patients treated with continuous ambulatory peritoneal dialysis (CAPD). Our observations were correlated to responses to treatment and with outcomes. The present study was carried out in 20 patients (8 males, 12 females) under CAPD. Peritonitis was diagnosed according to the criteria defined by the Ad Hoc Advisory Committee on Peritonitis Management. Peritoneal dialysate effluent (PDE) samples were collected from our patients, and lymphocyte subsets (CD2+, CD3+, CD3+/4+, CD3+/8+, CD3?/16+56+, CD4/CD8 ratio) were quantitated by using monoclonal antibodies. CD4/CD8 ratio was measured every day during peritonitis until the patients had completely recovered. The serial measurements of the CD4/CD8 ratio made in the PDE during peritonitis followed two patterns: the first pattern was characterized by a progressive increase in the CD4/CD8 ratio. The CD4/CD8 ratios on days 5, 6, and 7 were significantly higher than those on day 1 (P < 0.05). Overall, the patients who exhibited pattern 1 had favorable clinical courses. The second pattern was characterized by high initial CD4/CD8 ratios, which progressively decreased significantly (P < 0.05). This second pattern was associated with a delayed clinical response to treatment. Symptoms and signs of peritonitis persisted beyond 72 h. The pattern of the CD4/CD8 ratio in PDE may determine the outcome of peritonitis in CAPD patients.     

Donor‐Specific CD8+Foxp3+ T Cells Protect Skin Allografts and Facilitate Induction of Conventional CD4+Foxp3+ Regulatory T Cells

CD4⁺ regulatory T cells play a critical role in tolerance induction in transplantation. CD8⁺ suppressor T cells have also been shown to control alloimmune responses in preclinical and clinical models. However, the exact nature of the CD8⁺ suppressor T cells, their induction and mechanism of function in allogeneic transplantation remain elusive. In this study, we show that functionally suppressive, alloantigen‐specific CD8⁺Foxp3⁺ T cells can be induced and significantly expanded by stimulating naïve CD8⁺ T cells with donor dendritic cells in the presence of IL‐2, TGF‐β1 and retinoic acid. These CD8⁺Foxp3⁺ T cells express enhanced levels of CTLA‐4, CCR4 and CD103, inhibit the up‐regulation of costimulatory molecules on dendritic cells, and suppress CD4 and CD8 T cell proliferation and cytokine production in a donor‐specific and contact‐dependent manner. Importantly, upon adoptive transfer, the induced CD8⁺Foxp3⁺ T cells protect full MHC‐mismatched skin allografts. In vivo, the CD8⁺Foxp3⁺ T cells preferentially traffic to the graft draining lymph node where they induce conventional CD4⁺Foxp3⁺ T cells and concurrently suppress effector T cell expansion. We conclude that donor‐specific CD8⁺Foxp3⁺ suppressor T cells can be induced and exploited as an effective form of cell therapy for graft protection in transplantation.