Matthew R. Pipeling

CD4+CD25+Foxp3+ Tregs resolve experimental lung injury in mice and are present in humans with acute lung injury

Acute lung injury (ALI) is characterized by rapid alveolar injury, inflammation, cytokine induction, and neutrophil accumulation. Although early events in the pathogenesis of ALI have been defined, the mechanisms underlying resolution are unknown. As a model of ALI, we administered intratracheal (i.t.) LPS to mice and observed peak lung injury 4 days after the challenge, with resolution by day 10. Numbers of alveolar lymphocytes increased as injury resolved. To examine the role of lymphocytes in this response, lymphocyte-deficient Rag-1-/- and C57BL/6 WT mice were exposed to i.t. LPS. The extent of injury was similar between the groups of mice through day 4, but recovery was markedly impaired in the Rag-1-/- mice. Adoptive transfer studies revealed that infusion of CD4+CD25+Foxp3+ Tregs as late as 24 hours after i.t. LPS normalized resolution in Rag-1-/- mice. Similarly, Treg depletion in WT mice delayed recovery. Treg transfer into i.t. LPS-exposed Rag-1-/- mice also corrected the elevated levels of alveolar proinflammatory cytokines and increased the diminished levels of alveolar TGF-beta and neutrophil apoptosis. Mechanistically, Treg-mediated resolution of lung injury was abrogated by TGF-beta inhibition. Moreover, BAL of patients with ALI revealed dynamic changes in CD3+CD4+CD25hiCD127loFoxp3+ cells. These results indicate that Tregs modify innate immune responses during resolution of lung injury and suggest potential targets for treating ALI, for which there are no specific therapies currently available.

De novo donor-specific HLA antibodies are associated with early and high-grade bronchiolitis obliterans syndrome and death after lung transplantation

BACKGROUND The development of human leukocyte antigen (HLA) antibody responses has been associated with worse clinical outcomes, such as bronchiolitis obliterans syndrome (BOS) and death, in lung transplant recipients (LTRs). However, the role of donor-specific HLA antibody (DSA) responses as a risk factor for poor outcomes remains controversial. METHODS We prospectively screened 445 LTRs for DSA at our institution at the time of surveillance bronchoscopies for the first 2 years after transplantation between 2003 and 2008, and evaluated clinical outcomes. For this purpose, we used the combination of panel-reactive antibodies (PRA) by enzyme-linked immunosorbent assay (ELISA) and the Luminex single-antigen bead (SAB) assay (One Lambda, Canoga Park, CA). RESULTS We detected de novo DSA (dnDSA) in 58 of 445 (13%) LTRs in our cohort. Freedom from BOS was significantly reduced in LTRs with dnDSA versus those without dnDSA (p < 0.001). Using a Cox proportional hazards model, the development of dnDSA was associated with a significantly increased hazard ratio (HR = 6.59 [4.53 to 9.59]; p < 0.001) for BOS and high-grade BOS (Stage ≥ 2) (HR = 5.76 [3.48 to 9.52]; p < 0.001). Freedom from death was significantly reduced in LTRs with dnDSA (p < 0.001), including mortality attributable to BOS (HR = 9.86 [4.91 to 19.78]; p < 0.001). CONCLUSIONS Taken together, our findings provide evidence that dnDSA is associated with accelerated BOS kinetics and severity, as well as death due to BOS after lung transplantation. In addition, these data support regular monitoring for the development of dnDSA in LTRs and underscore the need for novel strategies to mitigate the increased risk of poor outcomes associated with dnDSA.

Comparisons of CD8+ T cells specific for human immunodeficiency virus, hepatitis C virus, and cytomegalovirus reveal differences in frequency, immunodominance, phenotype, and interleukin-2 responsiveness.

ABSTRACT To better understand the components of an effective immune response to human immunodeficiency virus (HIV), the CD8+ T-cell responses to HIV, hepatitis C virus (HCV), and cytomegalovirus (CMV) were compared with regard to frequency, immunodominance, phenotype, and interleukin-2 (IL-2) responsiveness. Responses were examined in rare patients exhibiting durable immune-mediated control over HIV, termed long-term nonprogressors (LTNP) or elite controllers, and patients with progressive HIV infection (progressors). The magnitude of the virus-specific CD8+ T-cell response targeting HIV, CMV, and HCV was not significantly different between LTNP and progressors, even though their capacity to proliferate to HIV antigens was preserved only in LTNP. In contrast to HIV-specific CD8+ T-cell responses of LTNP, HLA B5701-restricted responses within CMV pp65 were rare and did not dominate the total CMV-specific response. Virus-specific CD8+ T cells were predominantly CD27+45RO+ for HIV and CD27−45RA+ for CMV; however, these phenotypes were highly variable and heavily influenced by the degree of viremia. Although IL-2 induced significant expansions of CMV-specific CD8+ T cells in LTNP and progressors by increasing both the numbers of cells entering the proliferating pool and the number of divisions, the proliferative capacity of a significant proportion of HIV-specific CD8+ T cells was not restored with exogenous IL-2. These results suggest that immunodominance by HLA B5701-restricted cells is specific to HIV infection in LTNP and is not a feature of responses to other chronic viral infections. They also suggest that poor responsiveness to IL-2 is a property of HIV-specific CD8+ T cells of progressors that is not shared with responses to other viruses over which immunologic control is maintained.

Acute Kidney Injury Increases Mortality After Lung Transplantation

BACKGROUND Acute kidney injury requiring renal replacement therapy (RRT) is associated with increased mortality after cardiac surgery. Studies examining the impact of RRT after lung transplantation (LTx) are limited. We evaluated risk factors and outcomes associated with RRT after LTx. METHODS We retrospectively reviewed all LTx recipients in the United Network for Organ Sharing database. Preoperative renal function was stratified by glomerular filtration rate (GFR) as determined by the Modification of Diet in Renal Disease formula (strata: ≥90, 60 to 90, and <60 mL · min(-1) · 1.73 m(-2)). Primary outcomes were 30-day, 1-year, and 5-year survival and need for post-LTx RRT. Risk adjusted multivariable Cox proportional hazards regression examined mortality. A multivariable logistic regression model evaluated risk factors for RRT. RESULTS From 2001 to 2011, 12,108 patients underwent LTx. After LTx, 655 patients (5.51%) required RRT. Patients requiring post-LTx RRT had decreased survival at 30 days (96.7% versus 76.0%, p < 0.001), 1 year (85.5% versus 35.8%, p < 0.001), and 5 years (56.4% versus 20.0%, p < 0.001). These differences persisted on multivariable analysis at 30 days (hazard ratio [HR] 7.98 [6.16 to 10.33], p < 0.001), 1 year (HR 7.93 [6.84 to 9.19], p < 0.001), and 5 years (HR 5.39 [4.75 to 6.11], p < 0.001). Preoperative kidney function was an important predictor of post-LTx RRT for a GFR of 60 to 90 (odds ratio 1.42 [1.16 to 1.75], p = 0.001) and a GFR less than 60 (odds ratio 2.68 [2.07 to 3.46], p < 0.001]. High center volume was protective. CONCLUSIONS In the largest study to evaluate acute kidney injury after LTx, the incidence of RRT is 5.51%. The need for post-LTx RRT dramatically increases both short- and long-term mortality. Several variables, including preoperative renal function, are predictors of post-LTx RRT and could be used to identify transplant candidates at risk for acute kidney injury.

Successful treatment of Trichosporon mucoides infection with fluconazole in a heart and kidney transplant recipient.

Trichosporon species are an emerging cause of infection, particularly among transplant recipients. We report what we believe to be the first case of successful management of disseminated Trichosporon mucoides infection with orally administered fluconazole in a heart and kidney transplant recipient.

Primary Cytomegalovirus Phosphoprotein 65–Specific CD8+ T-Cell Responses and T-bet Levels Predict Immune Control During Early Chronic Infection in Lung Transplant Recipients

BACKGROUND Cytomegalovirus (CMV) remains an important pathogen in solid organ transplantation, particularly lung transplantation. Lung transplant recipients (LTRs) mismatched for CMV (donor positive/recipient negative [D(+)R(-)]) are at highest risk for active CMV infection and have increased mortality. However, the correlates of immune control during chronic CMV infection remain incompletely understood. METHODS We prospectively studied 22 D(+)R(-) LTRs during primary CMV infection and into chronic infection. Immune responses during primary infection were analyzed for association with viral relapse during early chronic infection. RESULTS Primary CMV infection was characterized by a striking induction of T-box transcription factor (T-bet) in CD8(+) T cells. CMV-specific effector CD8(+) T cells were found to be T-bet(+). After primary infection, 7 LTRs lacked immune control with relapsing viremia during early chronic infection. LTRs with relapsing viremia had poor induction of T-bet and low frequencies of phosphoprotein 65 (pp65)-specific CD8(+) effector T cells during primary infection. However, frequencies of IE1-specific CD8(+) effector T cells during primary infection were not associated with early relapsing viremia. CONCLUSIONS T-bet plays an important role in coordinating CD8(+) effector responses to CMV during primary infection. Moreover, CD8(+) T-bet induction and pp65-specific CD8(+) effector responses at the time of primary infection are important predictors of immune control of CMV during early chronic infection.

Differential CMV-Specific CD8+ Effector T Cell Responses in the Lung Allograft Predominate over the Blood during Human Primary Infection

Acquisition of T cell responses during primary CMV infection in lung transplant recipients (LTRs) appear critical for host defense and allograft durability, with increased mortality in donor+/recipient− (D+R−) individuals. In 15 D+R− LTRs studied, acute primary CMV infection was characterized by viremia in the presence or absence of pneumonitis, with viral loads higher in the lung airways/allograft compared with the blood. A striking influx of CD8+ T cells into the lung airways/allograft was observed, with inversion of the CD4+:CD8+ T cell ratio. De novo CMV-specific CD8+ effector frequencies in response to pooled peptides of pp65 were strikingly higher in lung mononuclear cells compared with the PBMC and predominated over IE1-specific responses and CD4+ effector responses in both compartments. The frequencies of pp65-specific cytokine responses were significantly higher in lung mononuclear cells compared with PBMC and demonstrated marked contraction with long-term persistence of effector memory CD8+ T cells in the lung airways following primary infection. CMV-tetramer+CD8+ T cells from PBMC were CD45RA− during viremia and transitioned to CD45RA+ following resolution. In contrast, CMV-specific CD8+ effectors in the lung airways/allograft maintained a CD45RA− phenotype during transition from acute into chronic infection. Together, these data reveal differential CMV-specific CD8+ effector frequencies, immunodominance, and polyfunctional cytokine responses predominating in the lung airways/allograft compared with the blood during acute primary infection. Moreover, we show intercompartmental phenotypic differences in CMV-specific memory responses during the transition to chronic infection.

Baculovirus-infected insect cells expressing peptide-MHC complexes elicit protective antitumor immunity

Evaluation of T cell responses to tumor- and pathogen-derived peptides in preclinical models is necessary to define the characteristics of efficacious peptide vaccines. We show in this study that vaccination with insect cells infected with baculoviruses expressing MHC class I linked to tumor peptide mimotopes results in expansion of functional peptide-specific CD8+ T cells that protect mice from tumor challenge. Specific peptide mimotopes selected from peptide-MHC libraries encoded by baculoviruses can be tested using this vaccine approach. Unlike other vaccine strategies, this vaccine has the following advantages: peptides that are difficult to solublize can be easily characterized, bona fide peptides without synthesis artifacts are presented, and additional adjuvants are not required to generate peptide-specific responses. Priming of antitumor responses occurs within 3 days of vaccination and is optimal 1 wk after a second injection. After vaccination, the Ag-specific T cell response is similar in animals primed with either soluble or membrane-bound Ag, and CD11c+ dendritic cells increase expression of maturation markers and stimulate proliferation of specific T cells ex vivo. Thus, the mechanism of Ag presentation induced by this vaccine is consistent with cross-priming by dendritic cells. This straightforward approach will facilitate future analyses of T cells elicited by peptide mimotopes.

T-bet:Eomes Balance, Effector Function, and Proliferation of Cytomegalovirus-Specific CD8+ T Cells during Primary Infection Differentiates the Capacity for Durable Immune Control

CMV remains an important opportunistic pathogen in solid organ transplantation, particularly in lung transplant recipients (LTRs). LTRs mismatched for CMV (donor+/recipient−; D+R−) are at high-risk for active CMV infection and increased mortality, however the immune correlates of viral control remain incompletely understood. We prospectively studied 23 D+R− LTRs during primary CMV infection to determine whether acute CD8+ T cell parameters differentiated the capacity for viral control in early chronic infection. T-box transcription factors expression patterns of T-bet > Eomesodermin (Eomes) differentiated LTR controllers from viremic relapsers and reciprocally correlated with granzyme B loading, and CMV phosphoprotein 65 (pp65)–specific CD8+IFN-γ+ and CD107a+ frequencies. LTR relapsers demonstrated reduced CD8+Ki67+ cells ex vivo and substantially impaired CD8+pp65-specific in vitro proliferative responses at 6 d, with concomitantly lower pp65-specific CD4+IL-2+ frequencies, as compared with LTR controllers. However, CMV-specific in vitro proliferative responses could be significantly rescued, most effectively with pp65 Ag and exogenous IL-2, resulting in an increased T-bet:Eomes balance, and enhanced effector function. Using class I CMV tetramers, we observed similar frequencies between relapsers and controllers, although reduced T-bet:Eomes balance in tetramer+ cells from relapsers, along with impaired CD8+ effector responses to tetramer-peptide restimulation. Taken together, these data show impaired CMV-specific CD8+ effector responses is not for complete lack of CMV-specific cells but rather underscores the importance of the T-bet:Eomes balance, with CMV-specific proliferation a key factor driving early T-bet expression and effector function in CD8+ T cells during primary infection and differentiating the capacity of high-risk LTRs to establish immune control during early chronic infection.

High-Quality CMV-Specific CD4+ Memory Is Enriched in the Lung Allograft and Is Associated With Mucosal Viral Control

The maintenance of CMV‐specific T cell memory in lung transplant recipients (LTRs) is critical for host defense and allograft durability, particularly in donor+/recipient− (D+R−) individuals who demonstrate increased mortality. We studied CD4+ and CD8+ CMV‐specific memory responses to phosphoprotein 65 (pp65) in a prospective cohort of 18 D+R− LTRs, from bronchoalveolar lavage (BAL)‐obtained lung mononuclear cells (LMNC) and PBMC. Unexpectedly, pp65‐specific CD4+ and CD8+ IFN‐γ memory responses from LMNC were similar, in contrast to persistent CD8+ predominance in PBMC. Unlike the pulmonary CD8+ predominance during acute primary infection, compartmental equalization occurred in the CMV‐specific CD8+ memory pool during chronic infection, whereas CMV‐specific CD4+ memory was enriched in the bronchoalveolar space. Moreover, CMV‐specific CD4+ memory T cells with multifunctional production of IFN‐γ, TNF‐α, IL‐2 and MIP‐1β were significantly increased in LMNCs, in contrast to similar intercompartmental CD8+ memory function. Moreover, the absolute number of CMV‐specific CD4+IFN‐γ+ memory cells in BAL was significantly increased in LTRs exhibiting viral control compared to those with CMV early antigen positivity. Collectively, these data demonstrate both preferential distribution and functional quality of CMV‐specific CD4+ memory in the lung allograft during chronic infection, and show an important association with CMV mucosal immunity and viral control.