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Introduction: Kidney transplants fail more often in Black than in non-Black (White, non-Black Hispanic, and Asian) recipients. We used the estimated physicochemical immunogenicity for polymorphic amino acids of donor/recipient HLAs to select weakly immunogenic kidney transplants for Black vs. White or non-Black patients., Methods: OPTN data for 65,040 donor/recipient pairs over a 20-year period were used to calculate the individual physicochemical immunogenicity by hydrophobic, electrostatic and amino acid mismatch scores (HMS, EMS, AMS) and graft-survival outcomes for Black vs. White or vs. non-Black recipients, using Kaplan-Meier survival and Cox regression analyses. Simulations for re-matching recipients with donors were based on race-adjusted HMS thresholds with clinically achievable allocations., Results: The retrospective median kidney graft survival was 12.0 years in Black vs. 18.6 years in White (6.6-year difference; p>0.001) and 18.4 years in non-Black (6.4-year difference; p>0.01) recipients. Only 0.7% of Blacks received transplants matched at HLA-A/B/DR/DQ (HMS=0) vs. 8.1% in Whites (p<0.001). Among fully matched Blacks (HMS=0), graft survival was 16.1-years and in well-matched Blacks (HMS ≤ 3.0) it was 14.0-years. Whites had 21.6-years survival at HMS ≤ 3.0 and 18.7-years at HMS ≤ 7.0 whereas non-Blacks had 22.0-year at HMS ≤ 3.0 and 18.7-year at HMS ≤ 7.0, confirming that higher HMS thresholds produced excellent survival. Simulation of ABO-compatible donor-recipient pairs using race-adjusted HMS thresholds identified weakly immunogenic matches at HMS=0 for 6.1% Blacks and 18.0% at HMS ≤ 3.0. Despite prioritizing Black patients, non-Black patients could be matched at the same level as in current allocation (47.0% vs 56.5%, at HMS ≤ 7.0)., Conclusions: Race-adjusted HMS (EMS, AMS)-based allocation increased the number of weakly immunogenic donors for Black patients, while still providing excellent options for non-Black recipients., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bekbolsynov, Mierzejewska, Khuder, Ekwenna, Rees, Green and Stepkowski.)

A growing body of evidence suggests that postural orthostatic tachycardia syndrome (POTS) may be an autoimmune disorder. We have reported in a previous manuscript that 89% of POTS patients ( n = 55) had elevations in G-protein-coupled adrenergic A1 receptor autoantibodies and 53% had elevations in muscarinic acetylcholine M4 receptor autoantibodies, as assessed by ELISA. Patients with autoimmune disorders have been reported with a variety of elevated cytokines and cytokines (such as rheumatoid arthritis); thus, we evaluated a limited number of cytokines/chemokines in POTS patients with elevated adrenergic and muscarinic receptor autoantibodies. We utilized the plasma of 34 patients from a previous study; all of the patients (100%) had autoantibodies against the A1 adrenergic receptor and 55.9% (19/34) had autoantibodies against the M4 muscarinic acetylcholine receptor. In particular, the plasma cytokine/chemokine levels were measured as biomarkers of inflammation by Quantibody ® technology (Raybiotech, Peachtree Corners, GA, USA). We also evaluated the platelet dense granule numbers, as these patients frequently complain of symptoms related to platelet dysfunction. Patients were predominantly young females who displayed a multitude of co-morbidities but generally reported viral-like symptoms preceding episodes of syncope. Eighty five percent (29/34) had platelet storage pool deficiency. Patients had elevations in five of ten cytokine/chemokines biomarkers (IL1β, IL21, TNFα, INFγ, and CD30), whereas two biomarkers had decreased levels (CD40L and RANTES). Our observations demonstrate that POTS patients known to have autoantibodies against the G-protein-coupled adrenergic A1 receptor have abnormal plasma concentrations of inflammatory cytokines.

We evaluated the impact of human leukocyte antigen (HLA) disparity (immunogenicity; IM) on long-term kidney allograft survival. The IM was quantified based on physicochemical properties of the polymorphic linear donor/recipient HLA amino acids (the Cambridge algorithm) as a hydrophobic, electrostatic, amino acid mismatch scores (HMS\AMS\EMS) or eplet mismatch (EpMM) load. High-resolution HLA-A/B/DRB1/DQB1 types were imputed to calculate HMS for primary/re-transplant recipients of deceased donor transplants. The multiple Cox regression showed the association of HMS with graft survival and other confounders. The HMS integer 0-10 scale showed the most survival benefit between HMS 0 and 3. The Kaplan-Meier analysis showed that: the HMS=0 group had 18.1-year median graft survival, a 5-year benefit over HMS>0 group; HMS ≤ 3.0 had 16.7-year graft survival, a 3.8-year better than HMS>3.0 group; and, HMS ≤ 7.8 had 14.3-year grafts survival, a 1.8-year improvement over HMS>7.8 group. Stratification based on EMS, AMS or EpMM produced similar results. Additionally, the importance of HLA-DR with/without -DQ IM for graft survival was shown. In our simulation of 1,000 random donor/recipient pairs, 75% with HMS>3.0 were re-matched into HMS ≤ 3.0 and the remaining 25% into HMS≥7.8: after re-matching, the 13.5 years graft survival would increase to 16.3 years. This approach matches donors to recipients with low/medium IM donors thus preventing transplants with high IM donors., (Copyright © 2020 Bekbolsynov, Mierzejewska, Borucka, Liwski, Greenshields, Breidenbach, Gehring, Leonard-Murali, Khuder, Rees, Green and Stepkowski.)

We examined what happened during a 6-year period to 1121 end-stage renal disease patients who registered with their willing/incompatible living donors for kidney exchanges with the Alliance for Paired Donation (APD). Of all patients, 65% were transplanted: 37% in kidney paired donation (APD-KPD, APD-other-KPD); 10% with compatible live donors (APD-LD); and 18% with deceased donors (APD-DD). The remaining patients were withdrawn (sick/died/others; 15%), or were still waiting (20%). For those patients with a cPRA 0-94%, 72% received a transplant. In contrast, only 49% of very highly sensitized (VHS; cPRA 95-100%) were transplanted. Of the VHS patients, 50% were transplanted by KPD/APD-LD while 50% benefited through prioritization of deceased donors in the modified kidney allocation system (KAS introduced in 2014). All APD transplanted groups had similar death-censored 4-year graft survivals as their relevant Organ Procurement and Transplantation Network (OPTN) groups. It is noteworthy that VHS graft and patient survival results were comparable to less sensitized and nonsensitized patients. All patients should be encouraged to search for compatible donors through different options. Expanding the donor pool through KPD and the new KAS of the OPTN increases the likelihood of transplantation for VHS patients., (© 2019 Steunstichting ESOT.)

Failure to convert computer-identified possible kidney paired donation (KPD) exchanges into transplants has prohibited KPD from reaching its full potential. This study analyzes the progress of exchanges in moving from "offers" to completed transplants. Offers were divided into individual segments called 1-way transplants in order to calculate success rates. From 2007 to 2014, the Alliance for Paired Donation performed 243 transplants, 31 in collaboration with other KPD registries and 194 independently. Sixty-one of 194 independent transplants (31.4%) occurred via cycles, while the remaining 133 (68.6%) resulted from nonsimultaneous extended altruistic donor (NEAD) chains. Thirteen of 35 (37.1%) NEAD chains with at least three NEAD segments accounted for 68% of chain transplants (8.6 tx/chain). The "offer" and 1-way success rates were 21.9 and 15.5%, respectively. Three reasons for failure were found that could be prospectively prevented by changes in protocol or software: positive laboratory crossmatch (28%), transplant center declined donor (17%) and pair transplanted outside APD (14%). Performing a root cause analysis on failures in moving from offer to transplant has allowed the APD to improve protocols and software. These changes have improved the success rate and the number of transplants performed per year., (© Copyright 2015 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Aims/hypothesis: T cells play a major role in the pathogenesis of type 1 diabetes, and there is great interest in developing curative immunotherapies targeting these cells. In this study, a monoclonal antibody (mAb) targeting the T cell receptor β-chain (TCRβ) was investigated for its ability to prevent and reverse disease in mouse models of diabetes., Methods: RIP-OVA(hi) (C57BL/6-Tg(Ins2-OVA)59Wehi/WehiJ) mice adoptively transferred with ovalbumin-specific T cells (an induced model of diabetes) and NOD mice (a spontaneous model of diabetes) were used to test anti-TCRβ mAb therapy as a means of preventing and reversing type 1 diabetes., Results: A single dose of anti-TCRβ completely prevented disease in RIP-OVA(hi) mice without inducing the release of inflammatory cytokines. Transient anti-TCRβ therapy prevented diabetes in 90% of NOD mice and reversed the disease after its onset in 73% of NOD mice. Long after the remission of type 1 diabetes, the anti-TCRβ treated mice were able to reject BALB/c skin allografts with normal kinetics while maintaining normoglycaemia. Treatment did not cause significant reductions in lymphocyte numbers in the spleen or pancreatic lymph nodes, but did result in a decreased percentage of chemokine receptor 9 (CCR9) positive, CD8(+) T cells. Notably, anti-TCRβ therapy increased the expression of programmed death 1 (PD-1) on the surface of the T cells; PD-1 expression is important for maintaining anti-TCRβ-induced self-tolerance, as type 1 diabetes recurs in mice following a blockade of PD-1 signalling., Conclusions/interpretation: Anti-TCRβ mAb is a safe and effective immunotherapy that results in reduced numbers of CCR9(+) T cells, an increased expression of PD-1 on T cells and the restoration of self-tolerance in NOD mice.

Background: Clinical application of islet transplantation to treat type 1 diabetes has been limited by islet allograft destruction by both allogeneic and autoimmune diabetogenic T-cell responses. The current study aims at determining whether an anti-T-cell receptor (TCR) monoclonal antibody (mAb) has potential as a novel and potent induction immunotherapy for islet transplantation., Methods: We have investigated the therapeutic efficacy and mechanisms of action of anti-TCR therapy in four different murine models, which comprise either allo- or autoimmune responses alone or both together., Results: T-cell response to islet allografts was potently abrogated by a brief treatment with an anti-TCRβ mAb (clone H57-597), resulting in long-term survival of BALB/c islet allografts in streptozotocin-induced diabetic B6 mice. Moreover, transient anti-TCR treatment permanently prevented BALB/c skin allograft rejection on Rag1 B6 recipients that were reconstituted with Foxp3 cell-depleted B6 splenocytes, but did not impair the reconstituted cells' ability to reject the later transplanted C3H skin allografts (transplanted at 120 days after BALB/c skin grafting). Transient anti-TCR treatment was also able to completely prevent diabetes onset in NOD.SCID.γc mice that were transferred with lymphocytes from diabetic NOD mice. Next, transient anti-TCR treatment significantly prolonged the survival of transplanted BALB/c islets in overtly diabetic NOD mice, which comprise both allogeneic and autoimmune diabetogenic T-cell responses to the transplanted islets., Conclusions: Overall, anti-TCR mAb induced peripheral tolerance to specific alloantigens even in the absence of Foxp3-expressing natural regulatory T cells. These findings reveal the potential for using TCR-targeting mAbs as induction immunotherapy for islet transplantation.

Optimal T cell activation and expansion require binding of the common gamma-chain (γc) cytokine Interleukin-2 (IL-2) to its cognate receptor that in turn engages a γc/Janus tyrosine kinase (Jak)3 signaling pathway. Because of its restricted expression by antigen-activated T cells and its obligatory role in promoting their survival and proliferation, IL-2 has been considered as a selective therapeutic target for preventing T cell mediated diseases. However, in order to further explore IL-2 targeted therapy, it is critical to precisely understand its role during early events of T cell activation. In this study, we delineate the role of IL-2 and other γc cytokines in promoting the survival of CD4 and CD8 T cells during early phases of priming. Under IL-2 inhibitory conditions (by neutralizing anti-IL-2 mAbs), the survival of activated CD8⁺ T cells was reduced, whereas CD4⁺ T cells remained much more resistant. These results correlated with reduced Bcl-2 expression, and mitochondrial membrane potential in CD8⁺ T cells in comparison to CD4⁺ T cells. However, using transwell co-culture assays we have found that CD4⁺ T cells could rescue the survival of CD8⁺ T cells even under IL-2 deprived conditions via secretion of soluble factors. A cytokine screen performed on CD8⁺ T cells cultured alone revealed that IL-21, another γc cytokine, was capable of rescuing their survival under IL-2 deprivation. Indeed, blocking the IL-21 signaling pathway along with IL-2 neutralization resulted in significantly reduced survival of both CD4⁺ and CD8⁺ T cells. Taken together, we have shown that under IL-2 deprivation conditions, IL-21 may act as the major survival factor promoting T cell immune responses. Thus, investigation of IL-2 targeted therapies may need to be revisited to consider blockade of the IL-21 signaling pathways as an adjunct to provide more effective control of T cell immune responses.

T cells play a major role in allograft rejection, which occurs after T cell activation by the engagement of several functional molecules to form an immune synapse with alloantigen presenting cells. In this study, the immune synapse was targeted using mAbs directed to the TCR beta-chain (TCRβ) and lymphocyte function-associated antigen-1 (LFA1) to induce long-term allograft survival. Evaluation of antigen-specific T cell responses was performed by adoptively transferring CFSE labeled transgenic OT-II cells into wild-type mice and providing OVA peptide by intravenous injection. Graft survival studies were performed in mice by transplanting BALB/c ear skins onto the flanks of C57BL/6 recipients. The anti-TCRβ plus anti-LFA1 mAb combination (but not either mAb alone) abrogated antigen-specific T cell responses invitro and invivo. Transient combination therapy with these agents resulted in significantly prolonged skin allograft survival in mice (51±10 days; p<0.01) when compared to treatment with either anti-TCRβ mAb (24±5 days) or anti-LFA1 mAb (19±3 days) alone or no treatment (10±1 days). When lymphoid tissues from these mice were analyzed at different times post-transplant, only those receiving the combination of anti-TCRβ and anti-LFA1 mAbs demonstrated long-lasting reductions in total T cell numbers, cellular and humoral anti-donor responses, and expression of CD3 on the surface of T cells. These results demonstrate that transient anti-TCRβ and anti-LFA1 mAb combination therapy abrogates antigen-reactive T cell responses with long-lasting effects that significantly prolong allograft survival.

Background: KRP203, a structural FTY720 analogue, has 5-fold greater selectivity for binding to sphingosine-1-phosphate receptor (S1PR) 1 (S1PR(1)) versus S1PR3 and 100-fold greater selectivity over S1PR(2) and S1PR(5). Although the immunoregulatory effects of FTY720 have been tested in clinical and experimental research, the therapeutic efficacy of KRP203 in allograft models remains elusive. In this study, we investigated the potential of KRP203 alone and in combination with intragraft injection of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) to induce islet allograft tolerance., Methods: BALB/c (H-2(d)) mice received transplants of fresh C57BL/10 (H-2(b)) islet allografts under the kidney capsule and were treated for 7 days with 0.3, 1.0, or 3.0 mg/kg KRP203 alone or in combination with intragraft-infused Tregs., Results: Untreated BALB/c mice acutely rejected C57BL/10 islet allografts at a mean survival time of 13.8 ± 2.7 days (n=5). A 7-day dosing of 0.3 or 1.0 mg/kg KRP203 produced long-term islet allograft survival (9200 days) in one of five and two of seven recipients, respectively. A 3 mg/kg KRP203 dose resulted in islet graft survival for more than 200 days in 5 of 12 recipients. Whereas recipients that received 500 allogeneic islets admixed with 5 x 10(5) - 7 x 10(5) Tregssurvived 83.6 ± 67.2 days, addition of transient 3 mg/kg KRP203 therapy induced prolonged drug-free graft survival (9200 days) in all recipients., Conclusions: A brief treatment with KRP203 significantly prolonged islet allograft survival, whereas additional intragraft delivery of Tregs induced tolerogenic effects selective to islet alloantigens.

The molecular mechanism of the extrathymic generation of adaptive, or inducible, CD4(+)Foxp3(+) regulatory T cells (iTregs) remains incompletely defined. We show that exposure of splenic CD4(+)CD25(+)Foxp3(-) cells to IL-2, but not other common γ-chain cytokines, resulted in Stat5 phosphorylation and induced Foxp3 expression in ∼10% of the cells. Thus, IL-2/Stat5 signaling may be critical for Foxp3 induction in peripheral CD4(+)CD25(+)Foxp3(-) iTreg precursors. In this study, to further define the role of IL-2 in the formation of iTreg precursors as well as their subsequent Foxp3 expression, we designed a two-step iTreg differentiation model. During the initial "conditioning" step, CD4(+)CD25(-)Foxp3(-) naive T cells were activated by TCR stimulation. Inhibition of IL-2 signaling via Jak3-Stat5 was required during this step to generate CD4(+)CD25(+)Foxp3(-) cells containing iTreg precursors. During the subsequent Foxp3-induction step driven by cytokines, IL-2 was the most potent cytokine to induce Foxp3 expression in these iTreg precursors. This two-step method generated a large number of iTregs with relatively stable expression of Foxp3, which were able to prevent CD4(+)CD45RB(high) cell-mediated colitis in Rag1(-/-) mice. In consideration of this information, whereas initial inhibition of IL-2 signaling upon T cell priming generates iTreg precursors, subsequent activation of IL-2 signaling in these precursors induces the expression of Foxp3. These findings advance the understanding of iTreg differentiation and may facilitate the therapeutic use of iTregs in immune disorders.

TCR specific antibodies may modulate the TCR engagement with antigen-MHC complexes, and in turn regulate in vivo T cell responses to alloantigens. Herein, we found that in vivo administration of mAbs specific for mouse TCRβ (H57-597), TCRα or CD3 promptly reduced the number of CD4(+) and CD8(+) T cells in normal mice, but H57-597 mAb most potently increased the frequency of CD4(+) Foxp3(+) Treg cells. When mice were injected with staphylococcal enterotoxin B (SEB) superantigen and H57-597 mAb, the expansion of SEB-reactive Vβ8(+) T cells was completely abrogated while SEB-nonreactive Vβ2(+) T cells remained unaffected. More importantly, transient H57-597 mAb treatment exerted long-lasting effect in preventing T cell responses to alloantigens, and produced long-term cardiac allograft survival (>100 days) in 10 out of 11 recipients. While Treg cells were involved in maintaining donor-specific long-term graft survival, T cell homeostasis recovered over time and immunity was retained against third party allografts. Moreover, transient H57-597 mAb treatment significantly prolonged survival of skin allografts in naïve recipients as well as heart allografts in skin-sensitized recipients. Thus, transient modulation of the TCRβ chain by H57-597 mAb exhibits potent, long-lasting therapeutic effects to control alloimmune responses., (© Copyright 2012 The American Society of Transplantation and the American Society of Transplant Surgeons.)

Maraviroc (MVC), a specific antagonist of CCR5 expressed on macrophages and activated T cells, may modulate inflammation and may be useful in patients with HIV infection. In this study we used nonhuman primates to examine the effect and mechanism of MVC alone or in combination with cyclosporine (CsA) to prolong cardiac allograft survivals. In an established rhesus monkey cardiac allograft model, recipients treated with MVC plus CsA showed significantly prolonged survival of heart allografts (>240 d, p < 0.001). These in vivo results in the MVC/CsA group correlated with delayed alloantibody response and markedly decreased graft infiltration by CCR5(+), CD4(+), CD8(+), and CD68(+) cells (p < 0.05), as compared with other groups. Furthermore, grafts from the MVC/CsA group had elevated numbers of alternatively activated macrophages (AAMs) and the expression of peroxisome proliferator-activated receptor γ (PPARγ). Blockade of PPARγ abrogated the prolonged allograft survival (median survival time, 45 d) and the upregulated AAMs in MVC/CsA-treated recipients. In conclusion, MVC/CsA protects cardiac allograft in primates and this effect is associated with generating AAMs through activation of the PPARγ nuclear receptor.

The benefits of immunotherapy by regulatory T (Treg) cells are unpredictable partially due to the uncertainty of their suppressive mechanism. In fact, various suppressive mechanisms have been proposed but each remains controversial. To better understand Treg-mediated suppression, we have investigated factors which may influence the suppressive effects. In an in vitro suppression assay, over-expression of anti-apoptotic Bcl2 enhancing survival of conventional T responder cells (Tconvs) did not subvert Treg-mediated suppression. In contrast, enhancing activation of Tconvs by increasing the potency of calcium signals completely abrogated Treg-mediated suppression. While Tregs were incapable of suppressing already activated Tconvs, they prevented expression of activation markers on naïve Tconvs during activation, thereby indicating that Tregs mediate suppression through controlling early activation stage. Interestingly, IL-2 deprivation or TGF-beta, two suppressive mechanisms, did not effectively inhibit Tconv activation and proliferation when applied alone. In contrast, IL-2 deprivation combined with TGF-beta suppressed Tconv activation as potently as Tregs. More importantly, in the transwell system, that separates Tregs from Tconvs, TGF-beta contributed to Treg suppression under IL-2 depriving condition. In conclusion, these two suppressive mechanisms acting in concert may be necessary to effectively restrain the early activation of Tconvs., (Copyright 2010 Elsevier B.V. All rights reserved.)

CD4(+)Foxp3(+) regulatory T (Treg) cells were shown to control all aspects of immune responses. How these Treg cells develop is not fully defined, especially in neonates during development of the immune system. We studied the induction of Treg cells from neonatal T cells with various TCR stimulatory conditions, because TCR stimulation is required for Treg cell generation. Independent of the types of TCR stimulus and without the addition of exogenous TGF-beta, up to 70% of neonatal CD4(+)Foxp3(-) T cells became CD4(+)Foxp3(+) Treg cells, whereas generally <10% of adult CD4(+)Foxp3(-) T cells became CD4(+)Foxp3(+) Treg cells under the same conditions. These neonatal Treg cells exert suppressive function and display relatively stable Foxp3 expression. Importantly, this ability of Treg cell generation gradually diminishes within 2 wk of birth. Consistent with in vitro findings, the in vivo i.p. injection of anti-CD3 mAb to stimulate T cells also resulted in a >3-fold increase in Treg cells in neonates but not in adults. Furthermore, neonatal or adult Foxp3(-) T cells were adoptively transferred into Rag1(-/-) mice. Twelve days later, the frequency of CD4(+)Foxp3(+) T cells converted from neonatal cells was 6-fold higher than that converted from adult cells. Taken together, neonatal CD4(+) T cells have an intrinsic "default" mechanism to become Treg cells in response to TCR stimulations. This finding provides intriguing implications about neonatal immunity, Treg cell generation, and tolerance establishment early in life.

Multiple activation signals (including antigen, co-stimulation, and cytokines) during T-cell priming affect the subsequent generation of memory T cells, whose survival is maintained by IL-7 and IL-15. Since the IL-7 receptor is highly expressed not only on the surface of memory T cells but also on naïve T cells, we propose that early exposure to IL-7 during priming of naïve T cells may promote their survival, and thus enhances the generation of memory cells. To test this hypothesis, TCR transgenic OT-II CD4(+) T cells were stimulated in vitro with OVA(323-339) peptide presented by syngeneic antigen-presenting cells (APCs). IL-7 or an IL-7 like cytokine, thymic stromal lymphopoietin (TSLP), was added at the initial 2-day cultivation stage. We found that a short exposure to IL-7 or TSLP during priming did not affect activation, proliferation, and glucose uptake by CD4(+) T cells compared to controls when examined on culture day 6. However, the same 6-day cultures showed that IL-7 (but not TSLP) significantly decreased the frequency of apoptotic CD4(+) T cells compared to controls. More importantly, an adoptive transfer of the 6-day primed OT-II T cells into CD45.1(+) congenic mice demonstrated that IL-7 (but not TSLP) elevated by 3- and 4-fold the number of transferred CD4(+) T cells in spleen (p<0.05) and lymph nodes (p<0.05), respectively, compared to controls. Almost all transferred CD4(+) T populations displayed phenotypes of effector (CD44(+)CD62L(-)) or central (CD44(+)CD62L(+)) memory T cells. We thus conclude that exposure of CD4(+) T cells to IL-7 during priming results in an increased frequency of CD4(+) memory T cells., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Background: We evaluated the impact of reduced nephron mass on nephrotoxicity by cyclosporine A (CsA) and/or sirolimus (SRL)., Methods: Renal function was tested in salt-depleted rats bearing two kidneys (2K), one kidney, or half a kidney (1/2K) and treated for 7 or 28 days with CsA (5 mg/kg) and/or SRL (0.8 mg/kg). We also measured the expression of aquaporin-2, sodium/phosphate cotransporter (NaPi)-2, paracellin-1, and kidney injury molecule (KIM)-1 by real-time polymerase chain reaction., Results: At 7 days in 2K, serum creatinine clearance (CrCl) was decreased only in CsA/SRL-treated group (P<0.05) compared with controls; in 1/2K, CrCl was decreased in all groups, but most dramatically in CsA/SRL group (P<0.05). Extended 28-day therapy worsened CrCl in all 1/2K groups (P<0.01). Although the expression of aquaporin-2, NaPi-2, and paracellin-1 mRNAs tended to increase in kidneys with a reduced nephron mass, NaPi-2 mRNA levels decreased in 1/2K rats exposed to CsA/SRL for 28 days (P<0.05). In contrast, low KIM-1 mRNA expression in control 2K rats increased fourfold in untreated 1/2K (P<0.05), and 50- to 200-fold in CsA/SRL-treated 1/2K (P=0.01)., Conclusions: Nephrotoxicity is significantly worsened by reduced nephron mass, which correlates with increased expression of KIM-1 and inhibited expression of NaPi-2.

The human immune system is a myriad of diverse cellular populations, each contributing to maintaining an effective and optimal immune response against infectious agents. It is important to maintain a "self-check" in the immune system so that responses do not go haywire, leading to the development of autoimmune diseases. Regulatory/suppressor T (Treg) cells are a specialized subpopulation of T cells that suppress the activation, expansion, and function of other T cells, thereby maintaining homeostasis through a fine balance between reactivity to foreign and self antigens. Tregs are characterized by surface expression of interleukin (IL)-2 receptor alpha chain (CD25) and intracellular expression of forkhead box protein P3 (FoxP3). There are at least two important functional populations of Treg cells, namely natural Treg (nTreg), which are continuously derived from the thymus, and induced Treg (iTreg), which are converted from naive T cells. The development and function of both nTreg and iTreg cells are regulated by several factors, such as antigen T-cell receptor, co-stimulatory receptors (i.e., cytotoxic T lymphocyte-associated antigen, or CTLA-4), and cytokines (IL-2, IL-10, and tumor growth factor-beta, or TGF-beta). In addition, the TGF-beta inhibitor ALK5, retinoid acid, and rapamycin influence the expansion of nTreg cells and the conversion of iTreg cells in vitro and in vivo. The heightening of Treg expansion may be harnessed to therapeutic methods for the treatment of autoimmune diseases and the induction of transplantation tolerance.

Agonists of the type 1 sphingosine-1-phosphate (S1P) receptor inhibit lymphocyte migration, causing their sequestration in lymphoid tissue. The S1P agonist FTY720 prolongs the survival of organ allografts and blocks T-cell mediated autoimmune diseases in experimental models; however, it is a non-selective agonist of four of the five S1P receptors. In this study female MRL/lpr mice, which develop an aggressive form of spontaneous autoimmune kidney disease, were treated with a more selective agonist of the type 1 receptor (KRP-203) or vehicle at 12 or 16 weeks of age. Eighty percent of the mice treated at 12 weeks, before the onset of visible disease, survived to the 24 weeks end point with decreased tubulointerstitial disease and significantly fewer infiltrating CD4(+) and CD8(+) T-cells. Only half of the control vehicle-treated mice survived. All of the mice treated at 16 weeks survived with reduced proteinuria. Mice in both groups had significant reductions in circulating lymphocytes. Mice receiving KRP-203 for 8-12 weeks had significant reductions in T-cells and consequently less adenopathy. Ex vivo treatment of lymphocytes from MRL/lpr mice with KRP-203 enhanced their apoptosis. Our study indicates that KRP-203 attenuates kidney injury in MRL/lpr mice, in part, by reducing T-cell infiltrates.

Maintaining T cell homeostasis is critical for normal immune response. Three sequential signals activate T cells, with signal 3 delivered by multiple cytokines that regulate cell proliferation, differentiation, and survival/death. Cytokines binding to their receptors engages two key molecular families, namely, Janus tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats). Among Stats, Stat3 is involved in the generation of T helper 17 (Th17) cells, regulation of dendritic cells, and acute inflammatory response. These aspects of Stat3 function are important for transplantation. We discuss Stat3's role in innate and adaptive immunity as well as its potential for therapeutic intervention.

Background: During pressure overload-induced hypertrophy, unloading-induced atrophy, and diabetes mellitus, the heart induces 'fetal' genes (e.g. myosin heavy chain beta; mhc beta)., Hypothesis: We propose that altered glucose homeostasis within the cardiomyocyte acts as a central mechanism for the regulation of gene expression in response to environmental stresses. The evidence is as follows., Methods and Results: Forced glucose uptake both ex vivo and in vivo results in mhc isoform switching. Restricting dietary glucose prevents mhc isoform switching in hearts of both GLUT1-Tg mice and rats subjected to pressure overload-induced hypertrophy. Thus, glucose availability correlates with mhc isoform switching under all conditions investigated. A potential mechanism by which glucose affects gene expression is through O-linked glycosylation of specific transcription factors. Glutamine:fructose-6-phosphate amidotransferase (GFAT) catalyzes the flux generating step in UDP-N-acetylglucosamine biosynthesis, the rate determining metabolite in protein glycosylation. Ascending aortic constriction increased intracellular levels of UDP-N-acetylglucosamine, and the expression of gfat2, but not gfat1, in the rat heart., Conclusions: Collectively, the results strongly suggest glucose-regulated gene expression in the heart, and the involvement of glucose metabolites in isoform switching of sarcomeric proteins characteristic for the fetal gene program.

We have previously shown that pretransplant donor lymphocyte infusion (DLI) together with transient depletion of CD4(+) T cells could induce permanent rat-to-mouse heart graft survival, whereas depleting CD4(+) T cells alone failed to do so. In this study, we investigated the mechanism leading to long-term xenograft survival. We found that peripheral CD4(+) T cells from DLI/anti-CD4-treated mice could mount rat heart graft rejection after adoptive transfer into B6 CD4(-/-) mice. Infusing donor-Ag-loaded mature dendritic cells (DCs) could break long-term cardiac xenograft survival in DLI/anti-CD4-treated mice. Interestingly, when the number and phenotype of graft-infiltrating cells were compared between anti-CD4- and DLI/anti-CD4-treated groups, we observed a significant increase in both the number and suppressive activity of alphabeta-TCR(+)CD3(+)CD4(-)CD8(-) double negative regulatory T cells and decrease in the numbers of CD4(+) and CD8(+) T cells in the xenografts of DLI/anti-CD4-treated mice. Moreover, there was a significant reduction in MHC class II-high DCs within the xenografts of DLI/anti-CD4-treated recipients. DCs isolated from the xenografts of anti-CD4- but not DLI/anti-CD4-treated recipients could stimulate CD4(+) T cell proliferation. Our data indicate that functional anti-donor T cells are present in the secondary lymphoid organs of the mice that permanently accepted cardiac xenografts. Their failure to reject xenografts is associated with an increase in double negative regulatory T cells as well as a reduction in Ag stimulation by DCs found within grafts. These findings suggest that local regulatory mechanisms need to be taken into account to control anti-xenograft T cell responses.

Regulation of T cell homeostasis is critical for maintaining normal immune function. An imbalance in T cell proliferation can result in disorders ranging from cancer and autoimmunity to immunodeficiencies. Full activation of T cells requires three sequential signals, where signal 3, which is delivered by multiple cytokines, regulates proliferation, differentiation, and survival/death. Signaling from cytokines through their receptors is primarily delivered by two molecular families, namely Janus tyrosine kinases (JAKs) and signal transducers and activators of transcription (STATs). Invaluable knowledge about JAKs and STATs has arisen from studies of mice made genetically deficient in these molecules, analyses of tumor models, and studies of expression patterns by proteomics/genomics, which all have begun to define the role of JAKs and STATs in survival versus apoptosis. These findings also have suggested ways in which JAKs and STATs may be manipulated for therapeutic intervention in lymphoid-derived diseases. This review seeks to focus on the role of JAK tyrosine kinases and STAT transcription factors in mediating the lymphocyte life cycle and how they might be manipulated for therapeutic applications.

Background: Previously we showed that pancreatic islets cultured for seven days in rotating bioreactors survived for >100 days in allogeneic recipients without immunosuppression. This survival coincided with almost complete elimination of "passenger" donor dendritic cells (DCs). Herein, we examined the necessity of DCs in the generation of CD4+ CD25+ T regulatory (Treg) cells., Methods: Allogeneic fresh islets or islets cultured for three days in bioreactors were transplanted to streptozotocin-induced diabetic Balb/c(stat4 -/-) as well as signal transducers and activators of transcription (Stat)4-deficient Balb/c(stat6 -/-) or Balb/c(stat4 -/-) mice. Some Balb/c recipients of fresh islet allografts were also treated with a tolerogenic protocol of anti-CD40 Ligand MR1 mAb and CTLA4Ig., Results: Islet allografts cultured for three days in bioreactors survived >100 days in all Balb/c(stat4 -/-) recipients and in 56% of Balb/c(stat6 -/-) recipients, but in none of the Balb/c recipients; the same recipients rejected fresh islet allografts. Purified T cells from long-term surviving Balb/c(stat4 -/-) recipients failed to transfer tolerance to SCID recipients of donor-type fresh islet allografts. In contrast, MR1/CTLA4Ig therapy induced tolerance to fresh islet allografts and their T cells adoptively transferred tolerance. When Balb/c or Balb/c(stat4 -/-) recipients of bioreactor-cultured islets were injected intravenously with immature syngeneic DCs, they became tolerant and developed potent alloantigen-specific CD4+ CD25+ Treg cells expressing Foxp3., Conclusion: Allogeneic islets depleted of donor DCs by culture in bioreactors have almost twofold better acceptance in Balb/c(stat4 -/-) than in Balb/c(stat6 -/-) mice, but lack Treg cells. Additional injection of host immature DCs improves tolerance in Balb/c and Balb/c(stat4 -/-) recipients by inducing potent CD4+ CD25+ Treg cells.

STATs are believed to play key roles in normal and abnormal cell function. In the present work, we investigated the role of STATs in an IL-2-responsive human lymphoblastic lymphoma-derived cell line, YT. Only STAT3 was found constitutively tyrosine phosphorylated, but not other STATs. Hyperactive STAT3 was not attributable to a pre-existing intermediate affinity IL-2R complex and/or hyperactive Jak activity. Depletion of STAT3 protein expression reduced tumor cell viability with protracted kinetics (72-96 h), while TUNEL assays demonstrated cell death occurred via apoptosis. Interestingly, depletion of STAT5 in this same tumor induced more pronounced cell death compared with STAT3 depletion (24 h). Although IL-2 was able to rescue STAT3-depleted cells from death, it could not compensate for the loss of STAT5. To determine the prosurvival function of STAT3 vs STAT5 within the same tumor model, genes were profiled in STAT3- or STAT5-depleted YT cells by apoptosis-specific microarrays. Several differentially expressed genes were identified. Interestingly, those genes involved in NF-kappaB regulation, such as TNFR-associated factors 2 and 5 and B cell leukemia/lymphoma 10, were readily decreased upon STAT5, but not STAT3, depletion as validated by quantitative RT-PCR. These results suggest that STAT5 and, to a lesser extent, hyperactive STAT3 provide preferential and critical cell survival signals for certain human lymphoid tumors, indicating that nonhyperactive STATs should be considered as therapeutic targets for abrogating tumorigenesis.

Full activation of T cells requires three sequential signals. Engagement by antigen presenting cells (APC) delivers signals 1/2, whereas signal 3 is delivered by multiple cytokines to regulate the immune homeostasis by influencing proliferation, differentiation, and survival/death. Signaling by cytokines acting through their receptors is delivered by two major molecular families, namely Janus tyrosine kinases (Jaks) and signal transducers and activators of transcription (Stats). Findings obtained from mice genetically deficient in Jaks and Stats suggest that these molecules may serve as therapeutic targets to prevent allograft rejection, induce transplantation tolerance, and inhibit autoimmune disease and lymphoid-derived tumors. This review describes the role of Jak tyrosine kinases and Stat transcription factors and their putative function in regulating T and B cell activity.

STATs play key roles in immune function. We examined the role of STAT5a/b in allograft rejection. STAT5a/b-deficient mice showed a 4-fold increased survival time of heart allografts (p < 0.01). Unlike wild type, purified STAT5a/b-/- T cells transferred to Rag1-/- recipients failed to mediate heart allograft rejection until supplemented with STAT5a/b-/- B cells. In vitro, STAT5a/b-/- T cells did not proliferate in response to Con A or alloantigens but entered apoptosis within 48 h (95%). Activated STAT5a/b-/- T cells showed increased expression of proapoptotic (caspases, DNA repair genes, TNF/TNFR-associated factor family genes) and decreased antiapoptotic mRNAs in microarrays, while Western blots confirmed reduced antiapoptotic Bcl-2 and elevated proapoptotic Bax protein expression. Interestingly, at 24 h postactivation, STAT5a/b+/+ and STAT5a/b-/- T cells produced similar levels of IL-2, IL-4, IL-10, and IFN-gamma mRNA; ELISPOT assay showed an equivalent number of IL-4- and IFN-gamma-producing T cells in both STAT5a/b+/+ and STAT5a/b-/- splenic populations. Sera from STAT5a/b+/+ and STAT5a/b-/- rejectors had donor-specific IgM, IgG1, IgG2a, and IgG2b Ab, while STAT5a/b deficiency had no impact on B cell survival or proliferation in response to LPS. Compared with allografts from STAT5a/b+/+ recipients, heart allografts from STAT5a/b-/- recipients had markedly reduced infiltration by CD4 and CD8 T cells but increased infiltration by B cells and dense endothelial deposition of C4d, a marker of humoral rejection. Thus, activated STAT5a/b-/- T cells produce cytokines prior to entering apoptosis, thereby promoting differentiation of B cells yielding donor-specific IgM and IgG Ab that mediate allograft rejection.

Background: The potential role of altered antibody responses as an effector protective mechanism to induce graft accommodation has been widely investigated in xenogeneic responses. Here we investigate the protective effects of antibody binding to vascular endothelium in a fully mismatched allogeneic model of heart transplantation., Methods: ACI recipients of WF cardiac grafts were treated either with allochimeric [alpha1h ]-RT1.A class I major histocompatibility complex (MHC) extracts (1 mg/rat, p.v. day 0) or high dose of CsA (10 mg/kg/day, p.o., day 0-6). Cardiac allografts were evaluated at 100 days posttransplant by immunohistology for evidence of chronic rejection and/or vascular accommodation. Activation of apoptotic or antiapoptotic mechanisms was verified by DNA fragmentation (TUNEL) analysis., Results: Allochimeric therapy resulted in inhibition of chronic rejection, absence of neointimal formation and induction of vascular accommodation of fully allogeneic WF hearts in ACI hosts. Such accommodation was evident by IgG and IgM vascular endothelial binding and marked reduction of DNA fragmentation. In contrast, CsA therapy resulted in marked neointimal proliferation, without evidence of vascular accommodation. Immunohistochemical analysis failed to demonstrate vascular endothelial antibody binding. Further, severe chronic rejection following CsA treatment was accompanied by marked DNA fragmentation., Conclusion: Alteration of humoral immunity induces vascular accommodation in allogeneic transplantation. Vascular accommodation is the underlying mechanism for inhibition allograft vasculopathy following allochimeric MHC class I therapy.

JAK3 is a cytoplasmic tyrosine kinase with limited tissue expression but is readily found in activated T cells. Patients lacking JAK3 are immune compromised, suggesting that JAK3 represents a therapeutic target for immunosuppression. Herein, we show that a Mannich base, NC1153, blocked IL-2-induced activation of JAK3 and its downstream substrates STAT5a/b more effectively than activation of the closely related prolactin-induced JAK2 or TNF-alpha-driven NF-kappaB. In addition, NC1153 failed to inhibit several other enzymes, including growth factor receptor tyrosine kinases, Src family members, and serine/threonine protein kinases. Although NC1153 inhibited proliferation of normal human T cells challenged with IL-2, IL-4, or IL-7, it did not block T cells void of JAK3. In vivo, a 14-day oral therapy with NC1153 significantly extended survival of MHC/non-MHC mismatched rat kidney allografts, whereas a 90-day therapy induced transplantation tolerance (>200 days). Although NC1153 acted synergistically with cyclosporin A (CsA) to prolong allograft survival, it was not nephrotoxic, myelotoxic, or lipotoxic and did not increase CsA-induced nephrotoxicity. In contrast to CsA, NC1153 was not metabolized by cytochrome P450 3A4. Thus, NC1153 prolongs allograft survival without several toxic effects associated with current immunosuppressive drugs.

Background: The addition of phosphorothioate (PS) groups to natural phosphodiester (PD) antisense oligodeoxynucleotides (oligo) prevents their in vivo hydrolysis by nucleases allowing an RNase-dependent elimination of targeted mRNA. To further improve oligo function 2'-methoxyethyl (ME) groups were attached to selected nucleotides at the 3'-end because ME groups block RNase activity., Methods/results: ME modification of PS- or PD/PS-oligo targeting human intracellular adhesion molecule (ICAM)-1 mRNA significantly increased the degree and duration of the in vitro inhibitory effects without compromising selectivity and specificity. A 7-day intravenous or oral therapy with rat ME/PS-modified ICAM-1 antisense oligo extended the survivals of kidney allografts. In addition, ME/PS-modified ICAM-1 antisense oligo reduced ischemic-reperfusion injury in kidneys, as measured by glomerular filtration rate, creatinine levels, and infiltration with leukocytes. Finally, a 14-day treatment with cyclosporine (CsA)-induced nephrotoxicity in syngeneic kidney transplants correlated with both increased ICAM-1 protein expression and infiltration with leukocytes. Graft perfusion and treatment of recipients with ICAM-1 antisense ME/PS-oligo alleviated the nephrotoxic effect and decreased ICAM-1 expression and leukocyte infiltration., Conclusions: ME/PS-modified ICAM-1 antisense oligo is very effective in inhibiting the ICAM-1-dependent mechanism of graft infiltration and tissue damage involved in allograft rejection, ischemic-reperfusion injury, and CsA-induced nephrotoxicity.

Binding of the P-, L-, and E-selectins to sialyl Lewis(x) (sLe(x)) retards circulating leukocytes, thereby facilitating their attachment to the blood vessels of allografts. Whether the selectin inhibitor bimosiamose (BIMO; C(46)H(54)O(16) . 0.25 H(2)O [867.4 molecular weight]) inhibits the rejection process of kidney allografts in a rat model was examined. Rat recipients acutely rejected kidney allografts at a mean survival time of 8.8 +/- 0.75 d. An intravenous 7-d infusion by osmotic pump of 2.5, 5, 10, or 20 mg/kg BIMO extended kidney allograft survival to 11.5 +/- 2.2 d (P < 0.03), 25.4 +/- 11.4 d (P < 0.006), 37.4 +/- 13.6 d (P < 0.001), and 39.8 +/- 34.5 d (P < 0.01), respectively. Combination of BIMO with cyclosporine produced synergistic interactions, as documented by the combination index (CI) values of 0.34 to 0.43 (CI <1 is synergistic; CI = 1 is additive; and CI >1 is antagonistic). Similarly, BIMO interacted synergistically with sirolimus (CI = 0.64) and FTY720 (CI = 0.22). While the mechanism of immunosuppression was being analyzed, decreased infiltration of CD4(+), CD8(+), and macrophages on day 7 after grafting was observed. Multiple cytokines were also expressed, including IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, TNF-alpha, and IFN-gamma in kidney allografts on days 3, 5, and 7 after grafting, as measured by a ribonuclease protection assay. Furthermore, at similar time points, BIMO treatment reduced intragraft expression of P-selectin glycoprotein ligand-1, CX(3)CL1, CCL19, CCL20, and CCL2. Thus, BIMO blocks allograft rejection by reduction of intragraft expression of cytokines and chemokines.

Background: When the immunosuppressants cyclosporine (CsA) and sirolimus (SRL) are co-administered to transplant patients, lower doses are used than when either drug is given alone. Since both drugs share similar transport and metabolic pathways, there is the potential for an interaction leading to unpredictable effects. Furthermore, both drugs affect the activity of cytochrome P450 3A1/2 (CYP3A1/2), the rat parallel to human CYP3A4, and the multidrug transporter P-glycoprotein (Pgp)., Methods: To clarify the role of metabolic enzymes and membrane transporters involved in the disposition of both drugs, we examined hepatic CYP3A1/2, Pgp, and multidrug resistance gene (mdr) mRNA during chronic therapy with CsA and SRL in salt-depleted rats. Specifically, rats were given intravenous doses of CsA 2.5 mg/kg and SRL 1 mg/kg, alone or in combination, for two weeks via constant rate intravenous infusion., Results: CsA treatment inhibited hepatic CYP3A1/2 protein expression, catalytic activity, and mRNA levels. SRL dosing suppressed CYP3A1/2 protein expression and catalytic activity, without affecting mRNA. With combined dosing, however, there was a much greater reduction. Hepatic Pgp protein levels were elevated after treatment with either drug alone, as well as with combined dosing. Compared to controls, there were significant increases in mdr1a and mdr1b mRNA levels in all treatment groups, with the combined drugs causing the greatest increase., Conclusions: Both CYP3A1/2 and Pgp participate in the disposition of CsA and SRL in rats. Changes in the individual activities of CYP3A1/2 and Pgp may contribute to an interaction between CsA and SRL resulting in unanticipated effects during chronic therapy.

Regulation of T- and B-lymphocyte survival and death is crucial for maintaining immune homeostasis. Unresponsiveness to death signals can result in lymphoproliferative disorders including cancer and autoimmunity, whereas lymphocytes hypersensitive to such signals can be manifested as immunodeficiencies. Often within these cells, cytokines and their receptors regulate the critical balance between life and death. It is becoming ever more apparent that within these effector cascades, Janus tyrosine kinases (Jak) and signal transducers and activators of transcription (Stat) act as key regulatory components. Invaluable knowledge about Jaks and Stats has arisen from mice made genetically deficient in these mqlecules, tumor models, and proteomics/genomics, which has begun to define their role in survival versus apoptosis. These findings have also suggested how Jaks and Stats might be manipulated for therapeutic intervention in lymphoid-derived diseases. This review seeks to focus on the role of Jak tyrosine kinases and Stat transcription factors in mediating the lymphocyte life cycle.

Background: Ten different highly polymorphic amino acids (AAs) are located in the alpha1 (alpha1h) and alpha2 (alpha2h) helical regions of the class I major histocompatibility complex RT1. An rat alloantigen. We examined the potential of alpha1h-RT1. An versus alpha2h-RT1. An polymorphic AAs to induce accelerated rejection or tolerance of heart allografts., Methods: The allochimeric alpha1h52-90n-RT1.Ac and alpha2h148-179n-RT1.Ac cDNAs were produced by the substitution of nucleotides encoding recipient RT1.Ac AAs for donor RT1. An AAs. Allochimeric and wild-type (WT)-RT1. An proteins were generated in an Escherichia coli expression system., Results: A single portal vein administration of 100 mug alpha1h52-90n-RT1.Ac protein in combination with a 7-day course of oral cyclosporine A (4 mg/kg) induced tolerance to Brown Norway (BN) (RT1n) heart allografts in PVG (RT1c) recipients more effectively than did WT-RT1. An protein; alpha2h148-179n-RT1.Ac protein was ineffective. However, subcutaneous injection of 100 mug WT-RT1. An (but neither alpha1h52-90n-RT1.Ac nor alpha2h148-179n-RT1.Ac) protein induced accelerated rejection of BN heart allografts. Untreated PVG recipients of BN heart allografts displayed activation of both interleukin (IL)-2- and interferon-gamma-producing T helper (Th) 1 cells and IL-4- and IL-10-producing Th2 cells on days 5, 7, and 14 postgrafting, as measured by an enzyme-linked immunospot assay. In contrast, in comparison with rejectors, tolerant recipients showed down-regulation of Th1 cells and up-regulation of Th2 cells on days 5, 7, 14, and 200 postgrafting. Histology of heart allografts showed that tolerant BN heart allografts had no evidence of acute or chronic rejection when examined on day 100 after transplantation., Conclusions: The poorly immunogenic alpha1h52-90n-RT1.Ac allochimeric protein induces tolerance by selective activation of regulatory Th2 cells.

Stat5a/b exhibits 96% homology and are required for normal immune function. The present studies examined Stat5a/b function in lymphoid cells by specific and simultaneous disruption of both proteins using novel phosphorothioate-2'-O-methoxyethyl antisense oligodeoxynucleotides (asODN). Efficient delivery was confirmed by the presence of fluorescent TAMRA-labeled ODN in >or=55 and 95% in human primary and tumor cell lines, respectively. Acute asODN administration reduced levels of Stat5a (90%) in 6 h, whereas Stat5b required nearly 48 h to attain the same inhibition, suggesting that the apparent turnover rate for Stat5a was 8-fold higher than that for Stat5b. Expression of the closely related Stat3 protein was unchanged after asODN treatment, however. Molecular ablation of Stat5a/b promoted apoptotic cell death in a significant population of primary PHA-activated T cells (72%) and lymphoid tumor cell line (e.g., YT; 74%) within 24 h, as assessed by 1) visualization of karyolytic nuclear degeneration and other generalized cytoarchitectural alterations, 2) enzymatic detection of TdT-positive DNA degradation, and 3) automated cytometric detection of annexin V translocation. Contrary to findings from Stat5a/b-null mice, cell cycle progression did not appear to be significantly affected. Interestingly, IL-2-insensitive and unprimed T cells and Jurkat cells remained mostly unaffected. Finally, evidence is provided that the cytotoxicity associated with Stat5a/b ablation may derive from activation of caspase-8, an initiator protease that contributes to apoptotic cell commitment. We propose that in lymphoid cells competent to activate Stat5a and Stat5b, both proteins preferentially mediate an antiapoptotic survival influence.

Although considerable progress has been achieved using immunosuppressive drugs that inhibit lymphocyte activation and T-cell cytokine signal transduction pathways, the widespread tissue distribution of the molecular targets exploited to date, calcineurin, mammalian target of rapamycin, and inosine monophosphate dehydrogenase, engenders a constellation of collateral toxicities. One strategy to develop new immunosuppressants seeks to identify targets that are critical for and specific to the adaptive immune response. Three approaches have been used to guide this enterprise; molecular design based on steric resemblance of the antagonist to the natural ligand; construction of complementary DNA oligonucleotides that hybridize with the leader sequence of messenger RNA encoding the synthesis of the specific target, thereby preventing production of that protein; and functional comparisons based on similar inhibitory profiles of candidate compounds and a probe that blocks the target nonselectively. Use of these 3 technologies has led to identification of antagonists blocking selectins, intercellular adhesion molecule-1, or Janus kinase 3, respectively. These lead compounds have been tested for their effects on the alloimmune response and/or the ischemia-reperfusion injuries.

Sirolimus (SRL; rapamycin) is a macrolide antibiotic, which modest anticandidal and tumoricidal activities were superseded by its immunosuppressive potential to block allograft rejection. The most intriguing biological characteristic of SRL emerged after demonstration of its potent synergism with cyclosporine (CsA). Naïve T cells, residing in the G(0) phase of the cell cycle, become activated by three signals. Signal 1 (T cell antigen receptor/alloantigen) and Signal 2 (CD28/B7) progress T cell to the early G(1) phase inducing production of interleukin-2 (IL-2) and other T cell growth factors (TGFs). Signal 3 (cytokine/cytokine receptor) initiate cell division and differentiation in the late G(1)/S phase. Whereas CsA binding to calcineurin blocks Signal 1/2, SRL binding to mammalian target of rapamycin (mTOR) blocks Signal 3. Our preclinical studies have established the in vivo principles of the effects exhibited by SRL alone on allograft survival, synergism between SRL and CsA as well as two drugs pharmacokinetic and pharmacodynamic interactions. In our experimental model, a 14-day i.v. continuous infusion of SRL by osmotic pump into rat recipients extended the survivals of heart allografts in a dose-dependent fashion. In comparison to untreated controls (MST of 6.3 +/- 0.5 days), 0.08 mg/kg SRL extended MST to 34.4 +/- 12.1 days, and 0.8 mg/kg to 74.1 +/- 20.2 days, with 6/18 allografts surviving for more than 100 days. Since almost identical results were produced by 10-fold higher SRL doses delivered by oral gavage, we estimated its bioavailability at 10%. Similarly, SRL prolonged the survivals of kidney, pancreas, and small bowel allografts in rats. At the same time large animal models cautioned about potential toxicities, namely intestinal vasculitis. The synergistic interactions of CsA and SRL may be explained by sequential effects in the early G(0)/G(1) versus late G1/S phases of cell cycle progression, respectively. The in vivo interaction of SRL with other immunosuppressive drugs was evaluated by the median effect analysis and the combination index (CI) values (CI = 1 shows additive, CI < 1, synergistic, and CI > 1, antagonistic, interactions). Oral SRL proved to be synergistic in both CsA-resistant mouse (CI = 0.4-1.5) and CsA-sensitive rat (CI = 0.3-0.6) models. The pharmacokinetic interactions of SRL and/or CsA were evaluated in rats for i.v. and oral formulations. Although low CsA and SRL i.v. doses did not affect each other levels, potent interaction was observed after oral gavage: CsA increased SRL levels by 2-11 folds; and, SRL increased CsA levels by 2-3-folds. Our results suggested that both pharmacodynamic and pharmacokinetic interactions contribute to the synergism between SRL and CsA. We also estimated the impact of CsA/SRL interaction on renal dysfunction, myelosuppression, and hyperlipidemia. Salt-depleted rats treated with SRL (0.4-6.4 mg/kg) and/or CsA (2.5-20 mg/kg) were examined for glomerular filtration rates (GFR), lipid levels, and bone marrow cellularity. CsA-induced kidney function deficiency was exacerbated by SRL. This exacerbation of renal dysfunction correlated with increased CsA levels in kidneys when combined with SRL. Furthermore, CsA potentiated SRL-mediated toxicities, namely myelosuppression and increased cholesterol. In conclusion, SRL therapy is synergistic with CsA but both drug levels should be carefully monitored to avoid toxic effects.

Relatively slow hematopoietic recovery after isolated bone marrow (I-BM) engraftment is probably caused by a disrupted microenvironment of stromal and stem cells. Thus, we compared the kinetics of hematopoietic recovery of lethally irradiated rats that received I-BM versus vascularized BM (V-BM). Total body irradiated (TBI; 8 Gy) Lewis (LEW; RT1(1)) rats were either injected IV with syngeneic sex-mismatched 80 x 10(6) I-BM or transplanted with 80 x 10(6) V-BM in orthotopic hind limb grafts. Ten days later, peripheral blood (PB) and mesenteric lymph nodes (MLN) of these recipients were examined for the presence of donor-derived hematopoietic cells with a panel of monoclonal antibodies by FACS. To detect male cells in sex-mismatched female recipients, PCR was performed using male Y chromosome primers. When examined in PB and MLN, recipients transplanted with V-BM displayed significantly faster recovery of leukocytes (CD43+), monocytes (CD14+), and T cells (CD5+) in comparison with I-BM recipients. In addition, only V-BM (but not I-BM) groups contained stroma-like male-positive cells in PB and MLN. Our results suggest that V-BM transplants provided superior hematopoietic recovery in comparison to I-BM transplants. We postulated that close proximity between stromal and stem cells in V-BM is essential for efficient repopulation with progenitors of different lines of leukocytes.

Signal transducer and activator of transcription (Stat)5a and Stat5b are critical for normal immune function. Progression of T cells through G(1)-S phase of cell cycle requires T cell receptor (TCR)- and/or cytokine-inducible tyrosine phosphorylation of Stat5a/b. Stat5a/b may also, in a cell-dependent manner, be constitutively or cytokine-inducibly phosphorylated on a Pro-Ser-Pro (PSP) motif located within the transcriptional activation domain. Phosphorylation of the PSP motif is needed for maximal transcriptional activation by Stat5, at least in certain promoter contexts. The basal and cytokine-inducible serine phosphorylation state of Stat5a/b has not been determined in T cells. Using primary human T cells and T lymphocytic cell lines coupled with novel phospho-specific antibodies to this conserved phosphoserine motif in Stat5a or Stat5b, we report that: Stat5a and Stat5b were unphosphorylated on the PSP motif under basal conditions and became markedly phosphorylated in response to several T cell growth factor stimuli, including interleukin (IL)-2, -7, -9, and -15 and phorbol ester 12-myristate 13-acetate but not TCR engagement; inducible Stat5a/b serine phosphorylation differed quantitatively and temporally; and Stat5a/b serine phosphorylation was, in contrast to inducible Stat3 serine phosphorylation, insensitive to inhibitors of mitogen-activated protein kinase, phosphatidylinositol-3 kinase, and mammalian target of rapamycin or deletion of Raf-A, -B, or -C by antisense oligonucleotides. We conclude that IL-2 family cytokines tightly control Stat5 serine phosphorylation through a kinase distinct from the Stat3 serine kinase.

Background: The failure of pancreatic islet allotransplants observed in almost all clinical attempts is related to poor initial islet function and allograft rejection. To remedy these problems we cultured islets in microgravity conditions to improve their function and to reduce their immunogenicity., Methods: Fresh mouse islets or mouse islets cultured in stationary dishes or microgravity bioreactors were transplanted to streptozotocin-induced diabetic mouse recipients., Results: Both allogeneic dish- or bioreactor-cultured islets survived more than 100 days compared with fresh allogeneic islets, which were rejected in less than 15 days. Islet titration studies revealed that 250 fresh or dish-cultured, but only 30 to 120 bioreactor-cultured, islets were necessary to produce euglycemia. Furthermore, glucose tolerance tests showed that bioreactor-cultured islets functioned better compared with fresh and dish-cultured islets on day 30 postgrafting. Immunostaining and transmission electron microscopy (TEM) analyses showed the gradual disappearance of dendritic cells in cultured islets compared with fresh islets. TEM revealed that the ultrastructure of islets from bioreactor, but not dish, appeared healthy and closely resembled fresh islets. Interestingly, TEM and scanning electron microscopy showed that only bioreactor-cultured islets developed unique and multiple nutritional channels between arrays of islet cells. TEM with colloidal lanthanum tracer revealed that only bioreactor islet cell cultures were devoid of tight junctional complexes, which may facilitate channel formation., Conclusion: Microgravity condition decreases immunogenicity and significantly improves the function of secretory cells.

Background: The various toxicities associated with the general immune suppression resulting from current clinical immunosuppressive therapies continue to plague transplant recipients as well as jeopardize allograft survival., Methods: The present study utilized allochimeric class I MHC antigens (alpha1hu70-77-RT1.Aa) bearing only four donor RT1.Au polymorphic amino acids (a.a.; His70, Val73, Asn74, and Asn77) superimposed on the recipient RT1.Aa background to induce transplantation tolerance in the rat cardiac transplant model., Results: Oral delivery of alpha1hu70-77-RT1.Aa protein alone (days 0-6) induced tolerance, as evidenced by inhibition of both acute and chronic rejection processes. Delivery of alpha1hu70-77-RT1.Aa with therapeutic doses of cyclosporine (CsA) also prevented chronic rejection, otherwise readily developed after treatment with CsA alone. A polymerase chain reaction (PCR)-based analysis showed that tolerant recipients had reduced numbers of interleukin (IL)-2/interferon (IFN)-gamma-producing T helper (Th)1 cells and elevated numbers of IL-4/IL-10-producing Th2 cells. Adoptive transfer experiments revealed that potent regulatory T cells mediated tolerance. The same T cells displayed diminished T cell receptor (TCR)-driven signaling via extracellular regulated kinase, AP-1, and NF-kappaB, as well as the common gamma-chain (gammac) cytokine-receptor-induced signaling by Janus kinase 3 (Jak3)/stimulators and activators of transcription Stat/5 pathways. Tolerance induction was prevented in vivo by inhibition of signal 2 by CTL4Ig or of signal 3 by either rapamycin, which disrupts the mammalian target of rapamycin, or AG490, which inhibits Jak3. Finally, partial or complete tyrosine phosphorylation of Zap70 was observed in alloantigen-specific T cell clones in response to tolerogenic versus immunogenic peptides, respectively., Conclusions: Tolerance induction by allochimeric proteins is achieved by partial TCR activation in the presence of signals 2 and 3, resulting in a skewed Th2 phenotype.

Janus kinase 3 (Jak3) is a cytoplasmic tyrosine (Tyr) kinase associated with the interleukin-2 (IL-2) receptor common gamma chain (gamma(c)) that is activated by multiple T-cell growth factors (TCGFs) such as IL-2, -4, and -7. Using human T cells, it was found that a recently discovered variant of the undecylprodigiosin family of antibiotics, PNU156804, previously shown to inhibit IL-2-induced cell proliferation, also blocks IL-2-mediated Jak3 auto-tyrosine phosphorylation, activation of Jak3 substrates signal transducers and activators of transcription (Stat) 5a and Stat5b, and extracellular regulated kinase 1 (Erk1) and Erk2 (p44/p42). Although PNU156804 displayed similar efficacy in blocking Jak3-dependent T-cell proliferation by IL-2, -4, -7, or -15, it was more than 2-fold less effective in blocking Jak2-mediated cell growth, its most homologous Jak family member. A 14-day alternate-day oral gavage with 40 to 120 mg/kg PNU156804 extended the survival of heart allografts in a dose-dependent fashion. In vivo, PNU156804 acted synergistically with the signal 1 inhibitor cyclosporine A (CsA) and additively with the signal 3 inhibitor rapamycin to block allograft rejection. It is concluded that inhibition of signal 3 alone by targeting Jak3 in combination with a signal 1 inhibitor provides a unique strategy to achieve potent immunosuppression.