IL‐17 Deficiency Attenuates Allograft Injury and Prolongs Survival in a Murine Model of Fully MHC‐Mismatched Renal Allograft Transplantation

IL‐17 is a pro‐inflammatory cytokine implicated in the pathogenesis of inflammatory and autoimmune diseases. However the role of IL‐17 in renal allograft rejection has not been fully explored. Here, we investigate the impact of IL‐17 in a fully MHC‐mismatched, life‐sustaining, murine model of kidney allograft rejection using IL‐17 deficient donors and recipients (IL‐17^?/? allografts). IL‐17^?/? allografts exhibited prolonged survival which was associated with reduced expression of the Th1 cytokine IFN‐γ and histological attenuation of acute and chronic allograft rejection, as compared to wild‐type allograft recipients. Results were confirmed in WT allograft recipients treated with an IL‐17 blocking antibody. Subsequent experiments using either donors or recipients deficient in IL‐17 showed a trend towards prolongation of survival only when recipients were IL‐17^?/?. Administration of a depleting anti‐CD25 antibody to IL‐17^?/? recipients abrogated the survival advantage conferred by IL‐17 deficiency, suggesting the involvement of a CD4⁺CD25⁺ T cell regulatory mechanism. Therefore, IL‐17 deficiency or neutralization was protective against the development of kidney allograft rejection, which may be mediated by impairment of Th1 responses and/or enhanced protection by Tregs.

     

Low‐Dose IL‐2 for In Vivo Expansion of CD4+ and CD8+ Regulatory T Cells in Nonhuman Primates

IL‐2 is a known potent T cell growth factor that amplifies lymphocyte responses in vivo. This capacity has led to the use of high‐dose IL‐2 to enhance T cell immunity in patients with AIDS or cancer. However, more recent studies have indicated that IL‐2 is also critical for the development and peripheral expansion of regulatory T cells (Tregs). In the current study, low‐dose IL‐2 (1 million IU/m² BSA/day) was administered to expand Tregs in vivo in naïve nonhuman primates. Our study demonstrated that low‐dose IL‐2 therapy significantly expanded peripheral blood CD4⁺ and CD8⁺ Tregs in vivo with limited expansion of non‐Treg cells. These expanded Tregs are mainly CD45RA^? Foxp3 ^high activated Tregs and demonstrated potent immunosuppressive function in vitro. The results of this preclinical study can serve as a basis to develop Treg immunotherapy, which has significant therapeutic potential in organ/cellular transplantation.     

Ex Vivo Expansion of Human Tregs by Rabbit ATG Is Dependent on Intact STAT3‐Signaling in CD4+ T Cells and Requires the Presence of Monocytes

The addition of low, nondepleting doses of rabbit antithymocyte globulin (ATG) to human peripheral blood mononuclear cells has been shown to expand functional CD4⁺CD25⁺FoxP3⁺ regulatory T cells (Tregs) in vitro. This report is the first to elucidate the exact cellular mechanisms of ATG‐mediated Treg expansion. CD4⁺ T cells require monocytes, but not other antigen presenting cell subsets, to be present in coculture to expand Tregs. However, T cells do not require direct cell–cell contact with monocytes, suggesting the importance of soluble factors. Moreover, ATG initially “reprograms” CD4⁺ T cells, but not monocytes, and induces STAT3 and STAT5 signaling in CD4⁺ cells. These reprogrammed CD4⁺ T cells subsequently secrete GM‐CSF and IL‐10 only in case of intact STAT3 signaling, which in turn promote the generation of tolerogenic CD14⁺CD11c⁺ dendritic cells characterized by enhanced IL‐10 and decreased IL‐12 production. Treg expansion following ATG treatment is accompanied by enhanced gene expression of both GM‐CSF and Bcl‐2, but not TGF‐β, in peripheral blood mononuclear cells. These results demonstrate that ex vivo expansion of human Tregs by ATG is due to its ability to reprogram CD4⁺ T cells in a STAT3‐dependent but TGF‐β‐independent manner, leading to the generation of monocyte‐derived dendritic cells with a tolerogenic cytokine profile.     

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.     

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.     

B cell–derived IL‐1β and IL‐6 drive T cell reconstitution following lymphoablation

Understanding the mechanisms of T cell homeostatic expansion is crucial for clinical applications of lymphoablative therapies. We previously established that T cell recovery in mouse heart allograft recipients treated with anti‐thymocyte globulin (mATG) critically depends on B cells and is mediated by B cell–derived soluble factors. B cell production of interleukin (IL)‐1β and IL‐6 is markedly upregulated after heart allotransplantation and lymphoablation. Neutralizing IL‐1β or IL‐6 with mAb or the use of recipients lacking mature IL‐1β, IL‐6, IL‐1R, MyD88, or IL‐6R impair CD4⁺ and CD8⁺ T cell recovery and significantly enhance the graft‐prolonging efficacy of lymphoablation. Adoptive co‐transfer experiments demonstrate a direct effect of IL‐6 but not IL‐1β on T lymphocytes. Furthermore, B cells incapable of IL‐1β or IL‐6 production have diminished capacity to mediate T cell reconstitution and initiate heart allograft rejection upon adoptive transfer into mATG treated B cell deficient recipients. These findings reveal the essential role of B cell–derived IL‐1β and IL‐6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation.     

Induction of porcine‐specific regulatory T cells with high specificity and expression of IL‐10 and TGF‐β1 using baboon‐derived tolerogenic dendritic cells

Background

Regulatory T cells (Treg) play an important role in maintenance of homeostasis in vivo. Treg application to alleviate allo‐organ rejection is being studied extensively. However, natural Treg (nTreg) expansion in vitro is laborious and expensive. Antigen‐specific Treg are more effective and require lower cell numbers than use of nTreg for immune control. The baboon, as a non‐human primate experimental animal model, is widely used in xenotransplantation research. An effective method to generate baboon xeno‐specific Treg would benefit research on immune tolerance in xenotransplantation using this model system.

Method

Baboon tolerogenic dendritic cells (tolDC) were generated in 3 days from monocytes isolated from baboon peripheral blood mononuclear cells in medium supplemented with anti‐inflammatory cytokines. After loading with porcine‐specific (PS) in vitro‐transcribed RNA (ivtRNA), tolDC were used to induce CD4⁺ T cells to become porcine‐specific Treg (PSTreg) in cocultures supplemented with IL‐2 and rapamycin for 10 days. Anti‐inflammatory and inflammatory cytokine expression was evaluated at the mRNA and protein levels in both baboon tolDC and PSTreg. Functional assays, suppression of activation markers on porcine‐specific effector T cells (PSTeff) and inhibition of PSTeff proliferation, were used to test PSTreg specificity.

Results

TolDC generated with this method exhibited a tolerogenic phenotype, expressed CCR7 and produced high levels of IL‐10 and TGF‐β1, whereas IL‐12p40 and IFN‐γ were not expressed. PSTreg were successfully generated in cocultures of CD4⁺ T cells and PS ivtRNA‐loaded tolDC. They exhibited a CD3⁺ CD4⁺ CD25⁺ CD127^low/? CD45RA^low Foxp3⁺ phenotype and were characterized by high expression of IL‐10 and TGF‐β1 mRNA and protein. They showed upregulated expression of EBI3 and GARP mRNA. PSTreg exhibited highly suppressive effects toward PSTeff, secreting high amounts of IL‐10 and TGF‐β1 cytokine upon interaction with PSTeff and suppressing IFN‐γ expression on PSTeff.

Conclusion

In this study, a fast 3‐day method to generate baboon‐derived tolDC is provided that allows subsequent induction of PSTreg displaying high porcine‐antigen specificity and expression of IL‐10 and TGF‐β1. Porcine‐specific baboon Treg can be used in porcine solid organ or cell xenotransplantation studies through adoptive cell transfer into host baboons.     

Antigen‐specific CD4+CD25+ T cells induced by locally expressed ICOS‐Ig: the role of Foxp3, Perforin,Granzyme B and IL‐10 ‐ an experimental study

We have previously reported that ICOS‐Ig expressed locally by a PIEC xenograft induces a perigraft cellular accumulation of CD4⁺CD25⁺Foxp3⁺ T cells and specific xenograft prolongation. In the present study we isolated and purified CD4⁺CD25⁺ T cells from ICOS‐Ig secreting PIEC grafts to examine their phenotype and mechanism of xenograft survival using knockout and mutant mice. CD4⁺CD25⁺ T cells isolated from xenografts secreting ICOS‐Ig were analysed by flow cytometry and gene expression by real‐time PCR. Regulatory function was examined by suppression of xenogeneic or allogeneic primed CD4 T cells in vivo. Graft prolongation was shown to be dependent on a pre‐existing Foxp3⁺ Treg, IL‐10, perforin and granzyme B. CD4⁺CD25⁺Foxp3⁺ T cells isolated from xenografts secreting ICOS‐Ig demonstrated a phenotype consistent with nTreg but with a higher expression of CD275 (ICOSL), expression of CD278 (ICOS) and MHC II and loss of CD73. Moreover, these cells were functional and specifically suppressed xenogeinic but not allogeneic primed T cells in vivo.     

CD4+CD28null T cells are not alloreactive unless stimulated by interleukin‐15

Proinflammatory, cytotoxic CD4⁺CD28^null T cells can be substantially expanded in patients with end‐stage renal disease. These cells have been associated with the risk for rejection, but their alloreactive potential is unknown. CD4⁺CD28^null T cells were stimulated with HLA‐mismatched antigen presenting cells in the absence/presence of exogenous cytokines. Alloreactive potential was evaluated based on proliferation, degranulation, cytotoxicity, and cytokine production. Further, their suppressive capacity was assessed by measuring inhibition of proliferating alloreactive CD28⁺ T cells. CD4⁺CD28^null T cells contained alloreactive (CD137⁺) T cells but did not proliferate in response to allogeneic stimulation, unless interleukin (IL)‐15 was added. However, they could proliferate on stimulation with cytomegalovirus antigen without exogenous cytokines. IL‐15 increased the frequency of proliferating alloreactive CD4⁺CD28^null T cells to 30.5% without inducing CD28 expression (P < .05). After allogeneic stimulation together with IL‐15 and IL‐21, frequency of degranulating CD107a⁺CD4⁺CD28^null T cells increased significantly from 0.6% to 5.8% (P < .001). Granzyme B and perforin positivity remained similar, but production of interferon‐γ and tumor necrosis factor‐α increased by the combination of IL‐15 and IL‐21 (P < .001 and P < .05, respectively). Finally, CD4⁺CD28^null T cells did not show significant suppression. Thus, CD4⁺CD28^null T cells represent a population with absent alloreactivity unless IL‐15 is present.     

CD4+ CD25+ regulatory T cells suppressed the indirect xenogeneic immune response mediated by porcine epithelial cell pulsed dendritic cells

Nishimura T, Onda M, Takao S. CD4⁺ CD25⁺ regulatory T cells suppressed the indirect xenogeneic immune response mediated by porcine epithelial cell pulsed dendritic cells. Xenotransplantation 2010; 17: 313–323. © 2010 John Wiley & Sons A/S. Abstract: Background: CD4⁺ CD25⁺ regulatory T cells have been reported to suppress T cell‐mediated xenogeneic immune responses. Although the direct T cell response to xenogeneic cells is important, the indirect xenogeneic immune response mediated by dendritic cells (DCs) is also likely involved in rejection. We have generated an in vitro indirect immune reaction model and evaluated the effect of CD4⁺ CD25⁺ regulatory T cells on this system. Methods: Human DCs were generated from peripheral blood and cultured with X‐ray‐irradiated porcine kidney epithelial cells. Porcine cell‐pulsed DCs were mixed with autologous CD4⁺ T cells, CD4⁺ CD25^? T cells and/or CD4⁺ CD25⁺ T cells. After 7 days of culture, T cell proliferation was measured. Results: The co‐culture of human DCs and X‐ray‐irradiated porcine epithelial cells resulted in observable DC phagocytic activity within 2 days. These porcine cell–pulsed DCs stimulated CD4⁺ T cell proliferation much more potently than unpulsed DCs or porcine cells. This proliferation was blocked by CTLA4‐Ig or an anti‐HLA‐DR antibody. CD4⁺ CD25⁺ regulatory T cells also suppressed CD4⁺ CD25^? T cell proliferation in response to porcine cell‐pulsed DCs. Conclusions: An in vitro model of the indirect xenogeneic immune response was established. Porcine cell‐pulsed DCs stimulated CD4⁺ T cells, and CD4⁺ CD25⁺ regulatory T cells suppressed this response.     

IL‐7 receptor heterogeneity as a mechanism for repertoire change during postdepletional homeostatic proliferation and its relation to costimulation blockade–resistant rejection

Kidney transplant patients treated with belatacept without depletional induction experience higher rates of acute rejection compared to patients treated with conventional immunosuppression. Costimulation blockade–resistant rejection (CoBRR) is associated with terminally differentiated T cells. Alemtuzumab induction and belatacept/sirolimus immunotherapy effectively prevent CoBRR. We hypothesized that cells in late phases of differentiation would be selectively less capable than more naive phenotypes of repopulating postdepletion, providing a potential mechanism by which lymphocyte depletion and repopulation could reduce the risk of CoBRR. Lymphocytes from 20 recipients undergoing alemtuzumab‐induced depletion and belatacept/sirolimus immunosuppression were studied longitudinally for markers of maturation (CCR7, CD45RA, CD57, PD1), recent thymic emigration (CD31), and the IL‐7 receptor‐α (IL‐7Rα). Serum was analyzed for IL‐7. Alemtuzumab induction produced profound lymphopenia followed by repopulation, during which naive IL‐7Rα⁺CD57^?PD1^? cells progressively became the predominant subset. This did not occur in a comparator group of 10 patients treated with conventional immunosuppression. Serum from depleted patients showed markedly elevated IL‐7 levels posttransplantation. Sorted CD57^?PD1^? cells demonstrated robust proliferation in response to IL‐7, whereas more differentiated cells proliferated poorly. These data suggest that differences in IL‐7‐dependent proliferation is one exploitable mechanism that distinguishes CoB‐sensitive and CoB‐resistant T cell populations to reduce the risk of CoBRR. (ClinicalTrials.gov ‐ NCT00565773.)     

Interleukin‐7 receptor blockade by an anti‐CD127 monoclonal antibody in nonhuman primate kidney transplantation

IL‐7 is an important cytokine for T cell lymphopoiesis. Blockade of the IL‐7 signaling pathway has been shown to induce long‐term graft survival or graft tolerance in murine transplant models through inhibiting T cell homeostasis and favoring immunoregulation. In this study, we assessed for the first time the effects of a blocking anti‐human cluster of differentiation 127 (CD127) mAb administered in combination with low‐dose tacrolimus or thymoglobulin in a life‐sustaining kidney allograft model in baboons. Contrary to our expectation, the addition of an anti‐CD127 mAb to the treatment protocols did not prolong graft survival compared to low‐dose tacrolimus alone or thymoglobulin alone. Anti‐CD127 mAb administration led to full CD127 receptor occupancy during the follow‐up period. However, all treated animals lost their kidney graft between 1 week and 2 weeks after transplantation. Unlike in rodents, in nonhuman primates, anti‐CD127 mAb treatment does not decrease the absolute numbers of lymphocyte and lymphocyte subsets and does not effectively inhibit postdepletional T cell proliferation and homeostasis, suggesting that IL‐7 is not a limiting factor for T cell homeostasis in primates.     

A Novel Xenogeneic Graft‐Versus‐Host Disease Model for Investigating the Pathological Role of Human CD4+ or CD8+ T Cells Using Immunodeficient NOG Mice

Graft‐versus‐host disease (GVHD) is a major complication of allogenic bone marrow transplantation and involves the infiltration of donor CD4⁺ and/or CD8⁺ T cells into various organs of the recipient. The pathological role of human CD4⁺ and CD8⁺ T cells in GVHD remains controversial. In this study, we established two novel xenogeneic (xeno)‐GVHD models. Human CD4⁺ or CD8⁺ T cells were purified from peripheral blood and were transplanted into immunodeficient NOD/Shi‐scid IL2rg^null (NOG) mice. Human CD8⁺ T cells did not induce major GVHD symptoms in conventional NOG mice. However, CD8⁺ T cells immediately proliferated and induced severe GVHD when transferred into NOG mice together with at least 0.5 × 10⁶ CD4⁺ T cells or into NOG human interleukin (IL)‐2 transgenic mice. Human CD4⁺ T cell–transplanted NOG mice developed skin inflammations including alopecia, epidermal hyperplasia, and neutrophilia. Pathogenic T helper (Th)17 cells accumulated in the skin of CD4⁺ T cell–transplanted NOG mice. Further, an anti‐human IL‐17 antibody (secukinumab) significantly suppressed these skin pathologies. These results indicate that pathogenic human Th17 cells induce cutaneous GVHD via IL‐17–dependent pathways. This study provides fundamental insights into the pathogenesis of xeno‐GVHD, and these humanized mouse models may be useful as preclinical tools for the prevention of GVHD.     

Association of Higher CD4+CD25highCD127low,FoxP3+, and IL‐2+ T Cell Frequencies Early After Lung Transplantation With Less Chronic Lung Allograft Dysfunction at Two Years

Regulatory T cells (Treg) can regulate alloantigens and may counteract chronic lung allograft dysfunction (CLAD) in lung transplantation. We analyzed Treg in peripheral blood prospectively and correlated percentages of subpopulations with the incidence of CLAD at 2 years. Among lung‐transplanted patients between January 2009 and July 2011, only patients with sufficient Treg measurements were included into the study. Tregs were measured immediately before lung transplantation, at 3 weeks and 3, 6, 12, and 24 months after transplantation and were defined as CD4⁺CD25^high T cells and further analyzed for CTLA4, CD127, FoxP3, and IL‐2 expressions. Between January 2009 and July 2011, 264 patients were transplanted at our institution. Among the 138 (52%) patients included into the study, 31 (22%) developed CLAD within 2 years after transplantation. As soon as 3 weeks after lung transplantation, a statistically significant positive association was detected between Treg frequencies and later absence of CLAD. At the multivariate analysis, increasing frequencies of CD4⁺CD25^highCD127^low, CD4⁺CD25^highFoxP3⁺ and CD4⁺CD25^highIL‐2⁺ T cells at 3 weeks after lung transplantation emerged as protective factors against development of CLAD at 2 years. In conclusion, higher frequencies of specific Treg subpopulations early after lung transplantation are protective against CLAD development.     

Ex-vivo expanded baboon CD4+ CD25 Hi Treg cells suppress baboon anti-pig T and B cell immune response

Singh AK, Seavey CN, Horvath KA, Mohiuddin MM. Ex‐vivo expanded baboon CD4⁺ CD25^Hi Treg cells suppress baboon anti‐pig T and B cell immune response. Xenotransplantation 2012; 19: 102–111. © 2012 John Wiley & Sons A/S. Abstract: Background: CD4⁺ CD25⁺ FoxP3⁺ regulatory T (Treg) cells play an important role in regulating immune responses. A very small number of Treg cells are present in peripheral blood and lymphoid organs, but due to their ability to suppress the immune response, they have a high potential for immunotherapy in clinics. Successful ex‐vivo expansion of naturally occurring CD4⁺ CD25⁺ T cells has been achieved after TCR stimulation in the presence of T cell growth factors. In this study, we evaluated the role of these Treg cells in suppressing proliferative response of baboon T and B cells to pig xenoantigens. Methods: Naturally occurring baboon CD4⁺ CD25⁺ regulatory T cells (nTreg) were sorted from peripheral blood and expanded in the presence of either anti‐CD3/CD28 beads or irradiated pig peripheral blood mononuclear cells with IL‐2. Treg cells were also enriched directly from CD4⁺ T cells cultured in the presence of rapamycin (0.1–10 nm ). Mixed lymphocyte culture and polyclonal B cell stimulation with ex‐vivo Treg cells were performed to assess the function of ex‐vivo expanded Treg cells. Results: The nTreg cells were expanded to more than 200‐fold in 4 weeks and retained all the nTreg cell phenotypic characteristics, including high levels of FoxP3 expression. 2‐fold increase in enrichment of CD4⁺ CD25⁺ FoxP3⁺ Treg cells from CD4⁺ cells was observed with rapamycin compared to cultures without rapamycin. The ex‐vivo expanded Treg cells obtained from both methods were able to suppress the baboon anti‐porcine xenogeneic T and B cell immune response in‐vitro efficiently (more than 90% suppression at 1 : 1 ratio of T regulatory cells: T effector cells), and their suppression potential was retained even at 1 : 256 ratio. However, freshly isolated nTreg cells had only 70% suppression at 1 : 1 ratio, and their suppressive ability was reduced to ≤50% at 1 : 16 ratio. Furthermore, we have found that ex‐vivo expanded Treg can also suppress the proliferation of B cells after polyclonal stimulation. Forty to 50 percent reduction in B cell proliferation was observed when ex‐vivo expanded Treg cells were added to the culture at a 1 : 1 ratio. The addition of CD4⁺ CD25^Neg cells however induced vigorous proliferation. Conclusion: Ex‐vivo expanded CD4⁺ CD25⁺ FoxP3⁺ Treg cells can be used to efficiently suppress xenogeneic immune responses by inhibiting T and B cell proliferation. These ex‐vivo expanded Treg cells may also be used with other immunosuppressive agents to overcome xenograft rejection in preclinical xenotransplantation models.     

A critical role for donor‐derived IL‐22 in cutaneous chronic GVHD

Graft‐versus‐host disease (GVHD) is the major cause of nonrelapse morbidity and mortality after allogeneic stem cell transplantation (allo‐SCT). Prevention and treatment of GVHD remain inadequate and commonly lead to end‐organ dysfunction and opportunistic infection. The role of interleukin (IL)‐17 and IL‐22 in GVHD remains uncertain, due to an apparent lack of lineage fidelity and variable and contextually determined protective and pathogenic effects. We demonstrate that donor T cell–derived IL‐22 significantly exacerbates cutaneous chronic GVHD and that IL‐22 is produced by highly inflammatory donor CD4⁺ T cells posttransplantation. IL‐22 and IL‐17A derive from both independent and overlapping lineages, defined as T helper (Th)22 and IL‐22⁺ Th17 cells. Donor Th22 and IL‐22⁺ Th17 cells share a similar IL‐6–dependent developmental pathway, and while Th22 cells arise independently of the IL‐22⁺Th17 lineage, IL‐17 signaling to donor Th22 directly promotes their development in allo‐SCT. Importantly, while both IL‐22 and IL‐17 mediate skin GVHD, Th17‐induced chronic GVHD can be attenuated by IL‐22 inhibition in preclinical systems. In the clinic, high levels of both IL‐17A and IL‐22 expression are present in the skin of patients with GVHD after allo‐SCT. Together, these data demonstrate a key role for donor‐derived IL‐22 in patients with chronic skin GVHD and confirm parallel but symbiotic developmental pathways of Th22 and Th17 differentiation.     

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.     

Enhanced Immunoprotective Effects by Anti‐IL‐17 Antibody Translates to Improved Skeletal Parameters Under Estrogen Deficiency Compared With Anti‐RANKL and Anti‐TNF‐α Antibodies

Activated T cell has a key role in the interaction between bone and immune system. T cells produce proinflammatory cytokines, including receptor activator of NF‐κB ligand (RANKL), tumor necrosis factor α (TNF‐α), and interleukin 17 (IL‐17), all of which augment osteoclastogenesis. RANKL and TNF‐α are targeted by inhibitors such as denosumab, a human monoclonal RANKL antibody, and infliximab, which neutralizes TNF‐α. IL‐17 is also an important mediator of bone loss, and an antibody against IL‐17 is undergoing phase II clinical trial for rheumatoid arthritis. Although there are a few studies showing suppression of Th17 cell differentiation and induction of regulatory T cells (Tregs) by infliximab, the effect of denosumab remains poorly understood. In this study, we investigated the effects of anti‐TNF‐α, anti‐RANKL, or anti‐IL‐17 antibody administration to estrogen‐deficient mice on CD4⁺ T‐cell proliferation, CD28 loss, Th17/Treg balance and B lymphopoesis, and finally, the translation of these immunomodulatory effects on skeletal parameters. Adult Balb/c mice were treated with anti‐RANKL/‐TNF‐α/‐IL‐17 subcutaneously, twice a week, postovariectomy (Ovx) for 4 weeks. Animals were then autopsied; bone marrow cells were collected for FACS and RNA analysis and serum collected for ELISA. Bones were dissected for static and dynamic histomorphometry studies. We observed that although anti‐RANKL and anti‐TNF‐α therapies had no effect on Ovx‐induced CD4⁺ T‐cell proliferation and B lymphopoesis, anti‐IL‐17 effectively suppressed both events with concomitant reversal of CD28 loss. Anti‐IL‐17 antibody reduced proinflammatory cytokine production and induced Tregs. All three antibodies restored trabecular microarchitecture with comparable efficacy; however, cortical bone parameters, bone biomechanical properties, and histomorphometry were best preserved by anti‐IL‐17 antibody, likely attributable to its inhibitory effect on osteoblast apoptosis and increased number of bone lining cells and Wnt10b expression. Based on the superior immunoprotective effects of anti‐IL‐17, which appears to translate to a better skeletal preservation, we propose beginning clinical trials using a humanized antibody against IL‐17 for treatment of postmenopausal osteoporosis. © 2014 American Society for Bone and Mineral Research.     

Allergic Conjunctivitis Renders CD4+ T Cells Resistant to T Regulatory Cells and Exacerbates Corneal Allograft Rejection

Allergic diseases rob corneal allografts of immune privilege and increase immune rejection. Corneal allograft rejection in BALB/c allergic hosts was analyzed using a short ragweed (SWR) pollen model of allergic conjunctivitis. Allergic conjunctivitis did not induce exaggerated T‐cell responses to donor C57BL/6 (B6) alloantigens or stimulate cytotoxic T lymphocyte (CTL) responses. Allergic conjunctivitis did affect T regulatory cells (Tregs) that support graft survival. Exogenous IL‐4, but not IL‐5 or IL‐13, prevented Treg suppression of CD4⁺ effector T cells isolated from naïve mice. However, mice with allergic conjunctivitis developed Tregs that suppressed CD4⁺ effector T‐cell proliferation. In addition, IL‐4 did not inhibit Treg suppression of IL‐4Rα^?/? CD4⁺ T‐cell responses, suggesting that IL‐4 rendered effector T cells resistant to Tregs. SRW‐sensitized IL‐4Rα^?/? mice displayed the same 50% graft survival as nonallergic WT mice, that was significantly less than the 100% rejection that occurred in allergic WT hosts, supporting the role of IL‐4 in the abrogation of immune privilege. Moreover, exacerbation of corneal allograft rejection in allergic mice was reversed by administering anti‐IL‐4 antibody. Thus, allergy‐induced exacerbation of corneal graft rejection is due to the production of IL‐4, which renders effector T cells resistant to Treg suppression of alloimmune responses.     

Evaluation of Alloreactivity in Kidney Transplant Recipients Treated with Antithymocyte Globulin Versus IL‐2 Receptor Blocker

Induction therapy is used in kidney transplantation to inhibit the activation of donor‐reactive T cells which are detrimental to transplant outcomes. The choice of induction therapy is decided based on perceived immunological risk rather than by direct measurement of donor T‐cell reactivity. We hypothesized that immune cellular alloreactivity pretransplantation can be quantified and that blocking versus depleting therapies have differential effects on the level of donor and third‐party cellular alloreactivity. We studied 31 kidney transplant recipients treated with either antithymocyte globulin (ATG) or an IL‐2 receptor blocker. We tested pre‐ and posttransplant peripheral blood cells by flow cytometry to characterize T‐cell populations and by IFN‐γ ELISPOT assays to assess the level of cellular alloreactivity. CD8⁺ T cells were more resistant to depletion by ATG than CD4⁺ T cells. Posttransplantation, frequencies of donor‐reactive T cells were markedly decreased in the ATG‐treated group but not in the IL‐2 receptor blocker group, whereas the frequencies of third‐party alloreactivity remained nearly equivalent. In conclusion, when ATG is used, marked and prolonged donor hyporesponsiveness with minimal effects on nondonor responses is observed. In contrast, induction with the IL‐2 receptor blocker is less effective at diminishing donor T‐cell reactivity.