Azithromycin Fails to Prevent Accelerated Airway Obliteration in T-bet-/- Mouse Lung Allograft Recipients.

BACKGROUND: Cellular and molecular mechanisms of acute and chronic lung allograft rejection have yet to be clearly defined, and obliterative bronchiolitis (OB) remains the primary limitation to survival in lung transplant recipients (LTRs). We have previously shown that T-bet-deficient recipients of full major histocompatibility complex (MHC)-mismatched, orthotopic left lung transplants develop accelerated obliterative airway disease (OAD) in the setting of acute cellular rejection characterized by robust alloimmune CD8+ interleukin (IL)-17 and interferon (IFN)-γ responses that are attenuated with neutralization of IL-17. Azithromycin has been shown to be beneficial in some LTRs with bronchiolitis obliterans syndrome/OB. Here, we evaluated the effects of azithromycin on rejection pathology and T-cell effector responses in T-bet-/- recipients of lung transplants. METHODS: Orthotopic left lung transplantation was performed in BALB/c → B6 wild type or BALB/c → B6 T-bet-/- strain combinations as previously described. Mice treated with azithromycin received 10 mg/kg or 50 mg/kg subcutaneously daily. Lung allograft histopathology was analyzed at day 10 or day 21 post-transplantation, and neutrophil staining for quantification was performed using anti-myeloperoxidase. Allograft mononuclear cells were isolated at day 10 for T-cell effector cytokine response assessment using flow cytometry. RESULTS: We show that while azithromycin significantly decreases lung allograft neutrophilia and CXCL1 levels and attenuates allospecific CD8+ IL-17 responses early post-transplantation, OAD persists in T-bet-deficient mice. CONCLUSIONS: Our results indicate that lung allograft neutrophilia is not essential for the development of OAD in this model and suggest allospecific T-cell responses that remain despite marked attenuation of CD8+ IL-17 are sufficient for obliterative airway inflammation and fibrosis.

Proteasome Inhibitor Carfilzomib-Based Therapy for Antibody-Mediated Rejection of the Pulmonary Allograft: Use and Short-Term Findings.

We present this observational study of lung transplant recipients (LTR) treated with carfilzomib (CFZ)-based therapy for antibody-mediated rejection (AMR) of the lung. Patients were considered responders to CFZ if complement-1q (C1q)-fixing ability of their immunodominant (ID) donor-specific anti-human leukocyte antibody (DSA) was suppressed after treatment. Treatment consisted of CFZ plus plasma exchange and immunoglobulins. Fourteen LTRs underwent CFZ for 20 ID DSA AMR. Ten (71.4%) of LTRs responded to CFZ. DSA IgG mean fluorescence intensity (MFI) fell from 7664 (IQR 3230-11 874) to 1878 (653-7791) after therapy (p = 0.001) and to 1400 (850-8287) 2 weeks later (p = 0.001). DSA C1q MFI fell from 3596 (IQR 714-14 405) to <30 after therapy (p = 0.01) and <30 2 weeks later (p = 0.02). Forced expiratory volume in 1s ( FEV1 ) fell from mean 2.11 L pre-AMR to 1.92 L at AMR (p = 0.04). FEV1 was unchanged after CFZ (1.91 L) and subsequently rose to a maximum of 2.13 L (p = 0.01). Mean forced expiratory flow during mid forced vital capacity (25-75) (FEF25-75 ) fell from mean 2.5 L pre-AMR to 1.95 L at AMR (p = 0.01). FEF25-75 rose after CFZ to 2.54 L and reached a maximum of 2.91 L (p = 0.01). Responders had less chronic lung allograft dysfunction or progression versus nonresponders (25% vs. 83%, p = 0.04). No deaths occurred within 120 days and 7 patients died post CFZ therapy of allograft failure. Larger prospective interventional studies are needed to further describe the benefit of CFZ-based therapy for pulmonary AMR.

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-1ß 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.

CD154 blockade abrogates allospecific responses and enhances CD4(+) regulatory T-cells in mouse orthotopic lung transplant.

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

CD154 deficiency uncouples allograft CD8+ T-cell effector function from proliferation and inhibits murine airway obliteration.

Obliterative bronchiolitis (OB) limits the long-term success of lung transplantation, while T-cell effector mechanisms in this process remain incompletely understood. Using the murine heterotopic tracheal transplant model of obliterative airway disease (OAD) to characterize airway allograft rejection, we previously reported an important role for CD8(+) T cells in OAD. Herein, we studied the role of CD154/CD40 costimulation in the regulation of allospecific CD8(+) T cells, as airway rejection has been reported to be CD154-dependent. Airway allografts from CD154(-/-) recipients had significantly lower day 28 OAD scores compared to wild-type (WT) recipients, and adoptive transfer of CD8(+) T cells from WT recipients, but not CD154(-/-) recipients, were capable of airway rejection in fresh CD154(-/-) allograft recipients. Intragraft CD8(+) T cells from CD154(-/-) mice showed similar expression of the surface markers CD69, CD62L(low) CD44(high) and PD-1, but markedly impaired IFN-gamma and TNF-alpha secretion and granzyme B expression versus WT controls. Unexpectedly, intragraft and systemic CD8(+) T cells from CD154(-/-) recipients demonstrated robust in vivo expansion similar to WT recipients, consistent with an uncoupling of proliferation from effector function. Together, these data suggest that a lack of CD154/CD40 costimulation results in ineffective allospecific priming of CD8(+) T cells required for murine OAD.

Differential effects of CD40 ligand/trimer stimulation on the ability of dendritic cells to replicate and transmit HIV infection: evidence for CC-chemokine-dependent and -independent mechanisms.

The role of exogenous stimulation of CD40 by CD40 ligand (CD40L) in dendritic cell (DC) maturation, CC-chemokine production, and CCR5 receptor expression was examined using a soluble trimeric CD40L agonist protein (CD40LT). Stimulation of monocyte-derived DCs with CD40LT enhanced the production of the CC-chemokines macrophage inflammatory protein (MIP)-1 alpha, MIP-1 beta, and RANTES and diminished surface expression of CCR5. Based on these findings, the functional role of CD40LT stimulation on the ability of DCs to replicate and transmit HIV viral infection was studied. The addition of CD40LT to cocultures of naive CD4+ T cells and autologous DCs (T/DC) infected with the macrophage-tropic isolate, HIVBaL, caused a striking reduction in reverse transcriptase (RT) activity after 10 and 14 days of culture. The addition of a mixture of Abs against CC-chemokines abrogated the decrease in RT activity, demonstrating that the inhibitory effect mediated by CD40LT was CC-chemokine-dependent. In contrast, the presence of CD40LT in T/DC cocultures infected with the T cell-tropic isolate, HIV IIIB, caused an increase in RT activity that was CC-chemokine-independent. Of note, CD40LT stimulation also inhibited RT activity in cultures containing macrophage-tropic virus (HIVBaL)-infected DC only. However, in contrast to the results seen in the T/DC cocultures, CD40LT stimulation inhibited RT activity in cultures of DCs alone in a CC-chemokine-independent manner. Together, these results show that CD40LT stimulation of DCs suppresses HIV replication and transmission to CD4+ T cells by two potentially different mechanisms.

Prostaglandin E2 and dexamethasone inhibit IL-12 receptor expression and IL-12 responsiveness.

Regulation of the factors governing IL-12R expression and IL-12 responsiveness has been shown to be important in the generation and stability of Th1- and Th2-type responses. In this regard, cytokines have been shown to have a prominent role in regulating IL-12R expression. In this study, the role that PGE2 and dexamethasone (DXM) have in regulating IL-12R expression was evaluated. Addition of PGE2 or DXM to human PBMCs stimulated with immobilized anti-CD3 plus IL-12 inhibited the production of IFN-gamma in a dose-responsive manner. Moreover, PBMCs stimulated with immobilized anti-CD3 in the presence of PGE2 or DXM for 3 days, washed extensively, and restimulated in the presence of IL-12 still did not produce IFN-gamma. This lack of IL-12 responsiveness from cells cultured in either PGE2 or DXM was correlated with diminished surface expression of IL-12Rbeta1, IL-12Rbeta2 mRNA expression, and IL-12 binding. Finally, the PGE2- and DXM-mediated inhibition of IL-12R expression was not affected significantly by addition of neutralizing Abs against either IL-4, IL-10, or TGF-beta. By contrast, addition of dibutyryl cAMP, 8-bromoadenosine 3:5 cAMP (8-Br-cAMP), or cholera toxin substantially reduced IL-12R expression, suggesting that PGE2 may be mediating its effects through enhancement of cAMP.

CD40 ligand/CD40 stimulation regulates the production of IFN-gamma from human peripheral blood mononuclear cells in an IL-12- and/or CD28-dependent manner.

CD40 ligand (CD40L)/CD40 costimulation is an important regulator of Th1 responses. Two mechanisms by which CD40L/CD40 stimulation may enhance IFN-gamma are via direct induction of IL-12 and augmentation of the expression of costimulatory molecules such as B7 from APCs. We examined the ability of CD40L/CD40 stimulation to regulate the production of IFN-gamma through IL-12 and/or CD28 costimulation from human PBMCs stimulated with T cell-specific stimuli. The roles of exogenous and endogenous CD40L/CD40 stimulation were evaluated using a trimeric soluble CD40L agonist (CD40T) and an anti-CD40L Ab, respectively. The presence of CD40T in cultures increased the production of IL-12 and IFN-gamma from PBMCs stimulated with varying amounts of PHA. The mechanism, however, by which CD40T enhanced IFN-gamma varied according to the level of T cell activation. Under maximal stimulatory conditions (PHA, 1/100), an IL-12-dependent pathway was dominant. At relatively low levels of T cell stimulation (PHA, 1/500 and 1/1000), however, an additional IL-12-independent CD28-dependent pathway was elucidated. We further studied the role of exogenous CD28 stimulation in regulating the production of IFN-gamma. The enhancement of IFN-gamma production induced by direct CD28 stimulation was primarily dependent on endogenous IL-12 or CD40L/CD40 stimulation. Together, these data suggest that the production of IFN-gamma involves a complex interaction between two interdependent, yet distinct, costimulatory pathways and provide evidence that CD40T may be an effective adjuvant for the enhancement of responses.

Patients with multidrug-resistant tuberculosis with low CD4+ T cell counts have impaired Th1 responses.

Multidrug-resistant tuberculosis (MDRTB) has emerged as a challenging clinical problem in both HIV-infected and -uninfected individuals. In this study, immune responses from HIV-negative patients with MDRTB were compared with those of healthy purified protein derivative (PPD)-positive and PPD-negative individuals. These responses were characterized by measuring the proliferation and cytokine production from PBMCs stimulated in vitro with Mycobacterium tuberculosis, PPD, or mitogens. MDRTB patients with CD4 counts >500/microl stimulated in vitro with M. tuberculosis had similar immune responses (proliferation, IFN-gamma, and IL-2 production) as the PPD-positive and -negative controls. By contrast, MDRTB patients with CD4 counts <500/microl had markedly deficient immune responses to similar stimuli. In these patients, IFN-gamma production could be restored by adding IL-12 to the in vitro cultures. IL-12 also caused a striking increase in the amount of IFN-gamma produced from PBMCs of both PPD-positive and -negative controls. The role of endogenous IL-12 production was also studied. Addition of anti-IL-12 to cultures resulted in a two- to eightfold decrease in IFN-gamma production in response to PHA stimulation. Inhibition of IFN-gamma was also observed when cells were stimulated by M. tuberculosis and PPD. Using Staphylococcus aureus Cowan strain as a mitogenic stimulus, IL-12 p70 was produced in similar amounts in all groups tested. TNF-alpha production was also assessed from cells stimulated by M. tuberculosis. Addition of IL-12 to the cultures did not cause a significant enhancement of TNF-alpha production. Last, production of IL-10 and IL-4 in response to M. tuberculosis and PHA, respectively, was not significantly different among all groups tested. These results suggest that patients with MDRTB tuberculosis with CD4 T cell counts <500/microl have impaired IFN-gamma and IL-2 responses and might benefit by adjunctive IL-12 therapy.

Cytokine interactions in human immunodeficiency virus-infected individuals: roles of interleukin (IL)-2, IL-12, and IL-15.

Cytokines have been shown to be powerful regulators of the immune response. In this study, we analyze the effect that the newly recognized cytokine interleukin (IL)-15 has on proliferation and cytokine induction using peripheral blood mononuclear cells (PBMCs) and purified CD4+ T cells from patients infected with human immunodeficiency virus (HIV) who are at various stages in their disease. We observed that IL-15 enhances the proliferative response in a dose-dependent manner from PBMCs of HIV-infected individuals when stimulated by polyclonal mitogen, tetanus toxoid, or HIV-specific antigen. The effects of exogenous IL-15 are substantially diminished by adding a neutralizing antibody to the beta chain of the IL-2 receptor. Moreover, the ability of IL-15 to increase proliferation is enhanced by the presence of endogenous IL-2 produced in the cultures. The effect that exogenous IL-15 had on IL-2, IL-4, and interferon (IFN)-gamma induction from PBMC's or CD4+ T cells in response to mitogen or tetanus toxoid was also examined. This was compared to the effect that exogenous IL-2 and IL-12 had under the same conditions. Addition of IL-2 or IL-15 to short-term in vitro cultures of either PBMCs or CD4+ T cells had little effect on IL-2, IL-4, or IFN-gamma production. By contrast, IL-12 caused substantial enhancement of both IL-2 and IFN-gamma production from these cultures. The role that endogenous cytokines have on IFN-gamma induction was also studied. Addition of a neutralizing antibody to the alpha chain of the IL-2 receptor or IL-12 to antigen stimulated cultures caused a striking decrease in IFN-gamma production. Neutralization of endogenous IL-15 also resulted in diminished IFN-gamma production from cultures stimulated with mitogen. IL-4 and IFN-gamma protein production by PBMCs and CD4+ T cells stimulated with mitogen was assessed to see if we could detect a specific bias of cytokine production. Small amounts of IL-4 were detected from CD4+ T cells but not PBMCs from most individuals tested. IFN-gamma and IL-2, however, were also produced from these same cultures. These results further elucidate the mechanism of cytokine regulation in HIV-infected individuals, and they provide evidence that IL-15 may be a useful immune modulator.