Prevention trumps treatment of antibody-mediated transplant rejection.

Belying the spectacular success of solid organ transplantation and improvements in immunosuppressive therapy is the reality that long-term graft survival rates remain relatively unchanged, in large part due to chronic and insidious alloantibody-mediated graft injury. Half of heart transplant recipients develop chronic rejection within 10 years - a daunting statistic, particularly for young patients expecting to achieve longevity by enduring the rigors of a transplant. The current immunosuppressive pharmacopeia is relatively ineffective in preventing late alloantibody-associated chronic rejection. In this issue of the JCI, Kelishadi et al. report that preemptive deletion of B cells prior to heart transplantation in cynomolgus monkeys, in addition to conventional posttransplant immunosuppressive therapy with cyclosporine, markedly attenuated not only acute graft rejection but also alloantibody elaboration and chronic graft rejection. The success of this preemptive strike implies a central role for B cells in graft rejection, and this approach may help to delay or prevent chronic rejection after solid organ transplantation.

Enhanced de novo alloantibody and antibody-mediated injury in rhesus macaques.

Chronic allograft rejection is a major impediment to long-term transplant success. Humoral immune responses to alloantigens are a growing clinical problem in transplantation, with mounting evidence associating alloantibodies with the development of chronic rejection. Nearly a third of transplant recipients develop de novo antibodies, for which no established therapies are effective at preventing or eliminating, highlighting the need for a nonhuman primate model of antibody-mediated rejection. In this report, we demonstrate that depletion using anti-CD3 immunotoxin (IT) combined with maintenance immunosuppression that included tacrolimus with or without alefacept reliably prolonged renal allograft survival in rhesus monkeys. In these animals, a preferential skewing toward CD4 repopulation and proliferation was observed, particularly with the addition of alefacept. Furthermore, alefacept-treated animals demonstrated increased alloantibody production (100%) and morphologic features of antibody-mediated injury. In vitro, alefacept was found to enhance CD4 effector memory T cell proliferation. In conclusion, alefacept administration after depletion and with tacrolimus promotes a CD4+memory T cell and alloantibody response, with morphologic changes reflecting antibody-mediated allograft injury. Early and consistent de novo alloantibody production with associated histological changes makes this nonhuman primate model an attractive candidate for evaluating targeted therapeutics.

Primary vascularization of the graft determines the immunodominance of murine minor H antigens during organ transplantation.

Grafts can be rejected even when matched for MHC because of differences in the minor histocompatibility Ags (mH-Ags). H4- and H60-derived epitopes are known as immunodominant mH-Ags in H2(b)-compatible BALB.B to C57BL/6 transplantation settings. Although multiple explanations have been provided to explain immunodominance of Ags, the role of vascularization of the graft is yet to be determined. In this study, we used heart (vascularized) and skin (nonvascularized) transplantations to determine the role of primary vascularization of the graft. A higher IFN-γ response toward H60 peptide occurs in heart recipients. In contrast, a higher IFN-γ response was generated against H4 peptide in skin transplant recipients. Peptide-loaded tetramer staining revealed a distinct antigenic hierarchy between heart and skin transplantation: H60-specific CD8(+) T cells were the most abundant after heart transplantation, whereas H4-specific CD8(+) T cells were more abundant after skin graft. Neither the tissue-specific distribution of mH-Ags nor the draining lymph node-derived dendritic cells correlated with the observed immunodominance. Interestingly, non-primarily vascularized cardiac allografts mimicked skin grafts in the observed immunodominance, and H60 immunodominance was observed in primarily vascularized skin grafts. However, T cell depletion from the BALB.B donor prior to cardiac allograft induces H4 immunodominance in vascularized cardiac allograft. Collectively, our data suggest that immediate transmigration of donor T cells via primary vascularization is responsible for the immunodominance of H60 mH-Ag in organ and tissue transplantation.

Interleukin-15 receptor blockade in non-human primate kidney transplantation.

BACKGROUND: Interleukin (IL)-15 is a chemotactic factor to T cells. It induces proliferation and promotes survival of activated T cells. IL-15 receptor blockade in mouse cardiac and islet allotransplant models has led to long-term engraftment and a regulatory T-cell environment. This study investigated the efficacy of IL-15 receptor blockade using Mut-IL-15/Fc in an outbred non-human primate model of renal allotransplantation. METHODS: Male cynomolgus macaque donor-recipient pairs were selected based on ABO typing, major histocompatibility complex class I typing, and carboxy-fluorescein diacetate succinimidyl ester-based mixed lymphocyte responses. Once animals were assigned to one of six treatment groups, they underwent renal transplantation and bilateral native nephrectomy. Serum creatinine level was monitored twice weekly and as indicated, and protocol biopsies were performed. Rejection was defined as a increase in serum creatinine to 1.5 mg/dL or higher and was confirmed histologically. Complete blood counts and flow cytometric analyses were performed periodically posttransplant; pharmacokinetic parameters of Mut-IL-15/Fc were assessed. RESULTS: Compared with control animals, Mut-IL-15/Fc-treated animals did not demonstrate increased graft survival despite adequate serum levels of Mut-IL-15/Fc. Flow cytometric analysis of white blood cell subgroups demonstrated a decrease in CD8 T-cell and natural killer cell numbers, although this did not reach statistical significance. Interestingly, two animals receiving Mut-IL-15/Fc developed infectious complications, but no infection was seen in control animals. Renal pathology varied widely. CONCLUSIONS: Peritransplant IL-15 receptor blockade does not prolong allograft survival in non-human primate renal transplantation; however, it reduces the number of CD8 T cells and natural killer cells in the peripheral blood.

CD4 enumeration technologies: a systematic review of test performance for determining eligibility for antiretroviral therapy.

BACKGROUND: Measurement of CD4+ T-lymphocytes (CD4) is a crucial parameter in the management of HIV patients, particularly in determining eligibility to initiate antiretroviral treatment (ART). A number of technologies exist for CD4 enumeration, with considerable variation in cost, complexity, and operational requirements. We conducted a systematic review of the performance of technologies for CD4 enumeration. METHODS AND FINDINGS: Studies were identified by searching electronic databases MEDLINE and EMBASE using a pre-defined search strategy. Data on test accuracy and precision included bias and limits of agreement with a reference standard, and misclassification probabilities around CD4 thresholds of 200 and 350 cells/μl over a clinically relevant range. The secondary outcome measure was test imprecision, expressed as % coefficient of variation. Thirty-two studies evaluating 15 CD4 technologies were included, of which less than half presented data on bias and misclassification compared to the same reference technology. At CD4 counts <350 cells/μl, bias ranged from -35.2 to +13.1 cells/μl while at counts >350 cells/μl, bias ranged from -70.7 to +47 cells/μl, compared to the BD FACSCount as a reference technology. Misclassification around the threshold of 350 cells/μl ranged from 1-29% for upward classification, resulting in under-treatment, and 7-68% for downward classification resulting in overtreatment. Less than half of these studies reported within laboratory precision or reproducibility of the CD4 values obtained. CONCLUSIONS: A wide range of bias and percent misclassification around treatment thresholds were reported on the CD4 enumeration technologies included in this review, with few studies reporting assay precision. The lack of standardised methodology on test evaluation, including the use of different reference standards, is a barrier to assessing relative assay performance and could hinder the introduction of new point-of-care assays in countries where they are most needed.

Characterization of CD4 and CD8 T cell responses in MuSK myasthenia gravis.

Muscle specific tyrosine kinase myasthenia gravis (MuSK MG) is a form of autoimmune MG that predominantly affects women and has unique clinical features, including prominent bulbar weakness, muscle atrophy, and excellent response to therapeutic plasma exchange. Patients with MuSK MG have predominantly IgG4 autoantibodies directed against MuSK on the postsynaptic muscle membrane. Lymphocyte functionality has not been reported in this condition. The goal of this study was to characterize T cell responses in patients with MuSK MG. Intracellular production of IFN-gamma, TNF-alpha, IL-2, IL-17, and IL-21 by CD4+ and CD8+ T cells was measured by polychromatic flow cytometry in peripheral blood samples from 11 Musk MG patients and 10 healthy controls. Only one MuSK MG patient was not receiving immunosuppressive therapy. Regulatory T cells (Treg) were also included in our analysis to determine if changes in T cell function were due to altered Treg frequencies. CD8+ T cells from MuSK MG patients had higher frequencies of polyfunctional responses than controls, and CD4+ T cells had higher IL-2, TNF-alpha, and IL-17. MuSK MG patients had a higher percentage of CD4+ T cells producing combinations of IFN-gamma/IL-2/TNF-gamma, TNF-alpha/IL-2, and IFN-gamma/TNF-alpha. Interestingly, Treg numbers and CD39 expression were not different from control values. MuSK MG patients had increased frequencies of Th1 and Th17 cytokines and were primed for polyfunctional proinflammatory responses that cannot be explained by a defect in CD39 expression or Treg number.

Impact of Leukocyte Function-Associated Antigen-1 Blockade on Endogenous Allospecific T Cells to Multiple Minor Histocompatibility Antigen Mismatched Cardiac Allograft.

BACKGROUND: Blocking leukocyte function-associated antigen (LFA)-1 in organ transplant recipients prolongs allograft survival. However, the precise mechanisms underlying the therapeutic potential of LFA-1 blockade in preventing chronic rejection are not fully elucidated. Cardiac allograft vasculopathy (CAV) is the preeminent cause of late cardiac allograft failure characterized histologically by concentric intimal hyperplasia. METHODS: Anti-LFA-1 monoclonal antibody was used in a multiple minor antigen-mismatched, BALB.B (H-2B) to C57BL/6 (H-2B), cardiac allograft model. Endogenous donor-specific CD8 T cells were tracked down using major histocompatibility complex multimers against the immunodominant H4, H7, H13, H28, and H60 minor Ags. RESULTS: The LFA-1 blockade prevented acute rejection and preserved palpable beating quality with reduced CD8 T-cell graft infiltration. Interestingly, less CD8 T cell infiltration was secondary to reduction of T-cell expansion rather than less trafficking. The LFA-1 blockade significantly suppressed the clonal expansion of minor histocompatibility antigen-specific CD8 T cells during the expansion and contraction phase. The CAV development was evaluated with morphometric analysis at postoperation day 100. The LFA-1 blockade profoundly attenuated neointimal hyperplasia (61.6 vs 23.8%; P < 0.05), CAV-affected vessel number (55.3 vs 15.9%; P < 0.05), and myocardial fibrosis (grade 3.29 vs 1.8; P < 0.05). Finally, short-term LFA-1 blockade promoted long-term donor-specific regulation, which resulted in attenuated transplant arteriosclerosis. CONCLUSIONS: Taken together, LFA-1 blockade inhibits initial endogenous alloreactive T-cell expansion and induces more regulation. Such a mechanism supports a pulse tolerance induction strategy with anti-LFA-1 rather than long-term treatment.

Leptin metabolically licenses T cells for activation to link nutrition and immunity.

Immune responses are highly energy-dependent processes. Activated T cells increase glucose uptake and aerobic glycolysis to survive and function. Malnutrition and starvation limit nutrients and are associated with immune deficiency and increased susceptibility to infection. Although it is clear that immunity is suppressed in times of nutrient stress, mechanisms that link systemic nutrition to T cell function are poorly understood. We show in this study that fasting leads to persistent defects in T cell activation and metabolism, as T cells from fasted animals had low glucose uptake and decreased ability to produce inflammatory cytokines, even when stimulated in nutrient-rich media. To explore the mechanism of this long-lasting T cell metabolic defect, we examined leptin, an adipokine reduced in fasting that regulates systemic metabolism and promotes effector T cell function. We show that leptin is essential for activated T cells to upregulate glucose uptake and metabolism. This effect was cell intrinsic and specific to activated effector T cells, as naive T cells and regulatory T cells did not require leptin for metabolic regulation. Importantly, either leptin addition to cultured T cells from fasted animals or leptin injections to fasting animals was sufficient to rescue both T cell metabolic and functional defects. Leptin-mediated metabolic regulation was critical, as transgenic expression of the glucose transporter Glut1 rescued cytokine production of T cells from fasted mice. Together, these data demonstrate that induction of T cell metabolism upon activation is dependent on systemic nutritional status, and leptin links adipocytes to metabolically license activated T cells in states of nutritional sufficiency.

Metabolic regulation of T lymphocytes.

T cell activation leads to dramatic shifts in cell metabolism to protect against pathogens and to orchestrate the action of other immune cells. Quiescent T cells require predominantly ATP-generating processes, whereas proliferating effector T cells require high metabolic flux through growth-promoting pathways. Further, functionally distinct T cell subsets require distinct energetic and biosynthetic pathways to support their specific functional needs. Pathways that control immune cell function and metabolism are intimately linked, and changes in cell metabolism at both the cell and system levels have been shown to enhance or suppress specific T cell functions. As a result of these findings, cell metabolism is now appreciated as a key regulator of T cell function specification and fate. This review discusses the role of cellular metabolism in T cell development, activation, differentiation, and function to highlight the clinical relevance and opportunities for therapeutic interventions that may be used to disrupt immune pathogenesis.

The liver kinase B1 is a central regulator of T cell development, activation, and metabolism.

T cell activation leads to engagement of cellular metabolic pathways necessary to support cell proliferation and function. However, our understanding of the signal transduction pathways that regulate metabolism and their impact on T cell function remains limited. The liver kinase B1 (LKB1) is a serine/threonine kinase that links cellular metabolism with cell growth and proliferation. In this study, we demonstrate that LKB1 is a critical regulator of T cell development, viability, activation, and metabolism. T cell-specific ablation of the gene that encodes LKB1 resulted in blocked thymocyte development and a reduction in peripheral T cells. LKB1-deficient T cells exhibited defects in cell proliferation and viability and altered glycolytic and lipid metabolism. Interestingly, loss of LKB1 promoted increased T cell activation and inflammatory cytokine production by both CD4(+) and CD8(+) T cells. Activation of the AMP-activated protein kinase (AMPK) was decreased in LKB1-deficient T cells. AMPK was found to mediate a subset of LKB1 functions in T lymphocytes, as mice lacking the α1 subunit of AMPK displayed similar defects in T cell activation, metabolism, and inflammatory cytokine production, but normal T cell development and peripheral T cell homeostasis. LKB1- and AMPKα1-deficient T cells each displayed elevated mammalian target of rapamycin complex 1 signaling and IFN-γ production that could be reversed by rapamycin treatment. Our data highlight a central role for LKB1 in T cell activation, viability, and metabolism and suggest that LKB1-AMPK signaling negatively regulates T cell effector function through regulation of mammalian target of rapamycin activity.

Cutting edge: distinct glycolytic and lipid oxidative metabolic programs are essential for effector and regulatory CD4+ T cell subsets.

Stimulated CD4(+) T lymphocytes can differentiate into effector T cell (Teff) or inducible regulatory T cell (Treg) subsets with specific immunological roles. We show that Teff and Treg require distinct metabolic programs to support these functions. Th1, Th2, and Th17 cells expressed high surface levels of the glucose transporter Glut1 and were highly glycolytic. Treg, in contrast, expressed low levels of Glut1 and had high lipid oxidation rates. Consistent with glycolysis and lipid oxidation promoting Teff and Treg, respectively, Teff were selectively increased in Glut1 transgenic mice and reliant on glucose metabolism, whereas Treg had activated AMP-activated protein kinase and were dependent on lipid oxidation. Importantly, AMP-activated protein kinase stimulation was sufficient to decrease Glut1 and increase Treg generation in an asthma model. These data demonstrate that CD4(+) T cell subsets require distinct metabolic programs that can be manipulated in vivo to control Treg and Teff development in inflammatory diseases.

Stereocomplexes Formed From Select Oligomers of Polymer d-lactic Acid (PDLA) and l-lactate May Inhibit Growth of Cancer Cells and Help Diagnose Aggressive Cancers-Applications of the Warburg Effect.

It is proposed that select oligomers of polymer d-lactic acid (PDLA) will form a stereocomplex with l-lactate in vivo, producing lactate deficiency in tumor cells. Those cancer cells that utilize transport of lactate to maintain electrical neutrality may cease to multiply or die because of lactate trapping, and those cancer cells that benefit from utilization of extracellular lactate may be impaired. Intracellular trapping of lactate produces a different physiology than inhibition of LDH because the cell loses the option of shuttling pyruvate to an alternative pathway to produce an anion. Conjugated with stains or fluorescent probes, PDLA oligomers may be an agent for the diagnosis of tissue lactate and possibly cell differentiation in biopsy specimens. Preliminary experimental evidence is presented confirming that PDLA in high concentrations is cytotoxic and that l-lactate forms a presumed stereocomplex with PDLA. Future work should be directed at isolation of biologically active oligomers of PDLA.

Preexisting compensatory amino acids compromise fitness costs of a HIV-1 T cell escape mutation.

BACKGROUND: Fitness costs and slower disease progression are associated with a cytolytic T lymphocyte (CTL) escape mutation T242N in Gag in HIV-1-infected individuals carrying HLA-B*57/5801 alleles. However, the impact of different context in diverse HIV-1 strains on the fitness costs due to the T242N mutation has not been well characterized. To better understand the extent of fitness costs of the T242N mutation and the repair of fitness loss through compensatory amino acids, we investigated its fitness impact in different transmitted/founder (T/F) viruses. RESULTS: The T242N mutation resulted in various levels of fitness loss in four different T/F viruses. However, the fitness costs were significantly compromised by preexisting compensatory amino acids in (Isoleucine at position 247) or outside (glutamine at position 219) the CTL epitope. Moreover, the transmitted T242N escape mutant in subject CH131 was as fit as the revertant N242T mutant and the elimination of the compensatory amino acid I247 in the T/F viral genome resulted in significant fitness cost, suggesting the fitness loss caused by the T242N mutation had been fully repaired in the donor at transmission. Analysis of the global circulating HIV-1 sequences in the Los Alamos HIV Sequence Database showed a high prevalence of compensatory amino acids for the T242N mutation and other T cell escape mutations. CONCLUSIONS: Our results show that the preexisting compensatory amino acids in the majority of circulating HIV-1 strains could significantly compromise the fitness loss due to CTL escape mutations and thus increase challenges for T cell based vaccines.

Isolation of HIV-1-neutralizing mucosal monoclonal antibodies from human colostrum.

BACKGROUND: Generation of potent anti-HIV antibody responses in mucosal compartments is a potential requirement of a transmission-blocking HIV vaccine. HIV-specific, functional antibody responses are present in breast milk, and these mucosal antibody responses may play a role in protection of the majority of HIV-exposed, breastfeeding infants. Therefore, characterization of HIV-specific antibodies produced by B cells in milk could guide the development of vaccines that elicit protective mucosal antibody responses. METHODS: We isolated B cells from colostrum of an HIV-infected lactating woman with a detectable neutralization response in milk and recombinantly produced and characterized the resulting HIV-1 Envelope (Env)-specific monoclonal antibodies (mAbs). RESULTS: The identified HIV-1 Env-specific colostrum mAbs, CH07 and CH08, represent two of the first mucosally-derived anti-HIV antibodies yet to be reported. Colostrum mAb CH07 is a highly-autoreactive, weakly-neutralizing gp140-specific mAb that binds to linear epitopes in the gp120 C5 region and gp41 fusion domain. In contrast, colostrum mAb CH08 is a nonpolyreactive CD4-inducible (CD4i) gp120-specific mAb with moderate breadth of neutralization. CONCLUSIONS: These novel HIV-neutralizing mAbs isolated from a mucosal compartment provide insight into the ability of mucosal B cell populations to produce functional anti-HIV antibodies that may contribute to protection against virus acquisition at mucosal surfaces.

Phenotypic characteristics of human type II alveolar epithelial cells suitable for antigen presentation to T lymphocytes.

Type II alveolar epithelial cells (AECII) are well known for their role in the innate immune system. More recently, it was proposed that they could play a role in the antigen presentation to T lymphocytes but contradictory results have been published both concerning their surface expressed molecules and the T lymphocyte responses in mixed lymphocyte cultures. The use of either AECII cell line or fresh cells could explain the observed discrepancies. Thus, this study aimed at defining the most relevant model of accessory antigen presenting cells by carefully comparing the two models for their expression of surface molecules necessary for efficient antigen presentation.