A Machine Learning-Driven Virtual Biopsy System For Kidney Transplant Patients.

In kidney transplantation, day-zero biopsies are used to assess organ quality and discriminate between donor-inherited lesions and those acquired post-transplantation. However, many centers do not perform such biopsies since they are invasive, costly and may delay the transplant procedure. We aim to generate a non-invasive virtual biopsy system using routinely collected donor parameters. Using 14,032 day-zero kidney biopsies from 17 international centers, we develop a virtual biopsy system. 11 basic donor parameters are used to predict four Banff kidney lesions: arteriosclerosis, arteriolar hyalinosis, interstitial fibrosis and tubular atrophy, and the percentage of renal sclerotic glomeruli. Six machine learning models are aggregated into an ensemble model. The virtual biopsy system shows good performance in the internal and external validation sets. We confirm the generalizability of the system in various scenarios. This system could assist physicians in assessing organ quality, optimizing allograft allocation together with discriminating between donor derived and acquired lesions post-transplantation.

The Banff 2022 Kidney Meeting Work Plan: Data-driven refinement of the Banff Classification for renal allografts.

The XVIth Banff Meeting for Allograft Pathology was held in Banff, Alberta, Canada, from September 19 to 23, 2022, as a joint meeting with the Canadian Society of Transplantation. In addition to a key focus on the impact of microvascular inflammation and biopsy-based transcript analysis on the Banff Classification, further sessions were devoted to other aspects of kidney transplant pathology, in particular T cell-mediated rejection, activity and chronicity indices, digital pathology, xenotransplantation, clinical trials, and surrogate endpoints. Although the output of these sessions has not led to any changes in the classification, the key role of Banff Working Groups in phrasing unanswered questions, and coordinating and disseminating results of investigations addressing these unanswered questions was emphasized. This paper summarizes the key Banff Meeting 2022 sessions not covered in the Banff Kidney Meeting 2022 Report paper and also provides an update on other Banff Working Group activities relevant to kidney allografts.

Development and Validation of a Multiclass Model Defining Molecular Archetypes of Kidney Transplant Rejection: A Large Cohort Study of the Banff Human Organ Transplant Gene Expression Panel.

Gene expression profiling from formalin-fixed paraffin-embedded (FFPE) renal allograft biopsies is a promising approach for feasibly providing a molecular diagnosis of rejection. However, large-scale studies evaluating the performance of models using NanoString platform data to define molecular archetypes of rejection are lacking. We tested a diverse retrospective cohort of over 1400 FFPE biopsy specimens, rescored according to Banff 2019 criteria and representing 10 of 11 United Network of Organ Sharing regions, using the Banff Human Organ Transplant panel from NanoString and developed a multiclass model from the gene expression data to assign relative probabilities of 4 molecular archetypes: No Rejection, Antibody-Mediated Rejection, T Cell-Mediated Rejection, and Mixed Rejection. Using Least Absolute Shrinkage and Selection Operator regularized regression with 10-fold cross-validation fitted to 1050 biopsies in the discovery cohort and technically validated on an additional 345 biopsies, our model achieved overall accuracy of 85% in the discovery cohort and 80% in the validation cohort, with ≥75% positive predictive value for each class, except for the Mixed Rejection class in the validation cohort (positive predictive value, 53%). This study represents the technical validation of the first model built from a large and diverse sample of diagnostic FFPE biopsy specimens to define and classify molecular archetypes of histologically defined diagnoses as derived from Banff Human Organ Transplant panel gene expression profiling data.

Self-Renewing CD8+ T-cell Abundance in Blood Associates with Response to Immunotherapy.

Treatment with immune checkpoint blockade (ICB) often fails to elicit durable antitumor immunity. Recent studies suggest that ICB does not restore potency to terminally dysfunctional T cells, but instead drives proliferation and differentiation of self-renewing progenitor T cells into fresh, effector-like T cells. Antitumor immunity catalyzed by ICB is characterized by mobilization of antitumor T cells in systemic circulation and tumor. To address whether abundance of self-renewing T cells in blood is associated with immunotherapy response, we used flow cytometry of peripheral blood from a cohort of patients with metastatic non-small cell lung cancer (NSCLC) treated with ICB. At baseline, expression of T-cell factor 1 (TCF1), a marker of self-renewing T cells, was detected at higher frequency in effector-memory (CCR7-) CD8+ T cells from patients who experienced durable clinical benefit compared to those with primary resistance to ICB. On-treatment blood samples from patients benefiting from ICB also exhibited a greater frequency of TCF1+CCR7-CD8+ T cells and higher proportions of TCF1 expression in treatment-expanded PD-1+CCR7-CD8+ T cells. The observed correlation of TCF1 frequency in CCR7-CD8+ T cells and response to ICB suggests that broader examination of self-renewing T-cell abundance in blood will determine its potential as a noninvasive, predictive biomarker of response and resistance to immunotherapy.

High-speed light-sheet microscopy for the in-situ acquisition of volumetric histological images of living tissue.

Histological examinations typically require the excision of tissue, followed by its fixation, slicing, staining, mounting and imaging, with timeframes ranging from minutes to days. This process may remove functional tissue, may miss abnormalities through under-sampling, prevents rapid decision-making, and increases costs. Here, we report the feasibility of microscopes based on swept confocally aligned planar excitation technology for the volumetric histological imaging of intact living tissue in real time. The systems' single-objective, light-sheet geometry and 3D imaging speeds enable roving image acquisition, which combined with 3D stitching permits the contiguous analysis of large tissue areas, as well as the dynamic assessment of tissue perfusion and function. Implemented in benchtop and miniaturized form factors, the microscopes also have high sensitivity, even for weak intrinsic fluorescence, allowing for the label-free imaging of diagnostically relevant histoarchitectural structures, as we show for pancreatic disease in living mice, for chronic kidney disease in fresh human kidney tissues, and for oral mucosa in a healthy volunteer. Miniaturized high-speed light-sheet microscopes for in-situ volumetric histological imaging may facilitate the point-of-care detection of diverse cellular-level biomarkers.

Association between procurement biopsy findings and deceased donor kidney outcomes: a paired kidney analysis.

Unfavourable procurement biopsy findings are the most common reason for deceased donor kidney discard in the United States. We sought to assess the association between biopsy findings and post-transplant outcomes when donor characteristics are accounted for. We used registry data to identify 1566 deceased donors of 3132 transplanted kidneys (2015-2020) with discordant right/left procurement biopsy classification and performed time-to-event analyses to determine the association between optimal histology and hazard of death-censored graft failure or death. We then repeated all analyses using a local cohort of 147 donors of kidney pairs with detailed procurement histology data available (2006-2016). Among transplanted kidney pairs in the national cohort, there were no significant differences in incidence of delayed graft function or primary nonfunction. Time to death-censored graft failure was not significantly different between recipients of optimal versus suboptimal kidneys. Results were similar in analyses using the local cohort. Regarding recipient survival, analysis of the national, but not local, cohort showed optimal kidneys were associated with a lower hazard of death (adjusted HR 0.68, 95% CI 0.52-0.90, P = 0.006). In conclusion, in a large national cohort of deceased donor kidney pairs with discordant right/left procurement biopsy findings, we found no association between histology and death-censored graft survival.

The clinical significance of receiving a kidney allograft from deceased donor with chronic histologic changes.

Allograft survival of deceased donor kidneys with suboptimal histology (DRTx/suboptimal histology: >10% glomerulosclerosis, >10% tubulointerstitial scarring, or >mild vascular sclerosis) is inferior to both DRTx with optimal histology (DRTx/optimal histology) and living donor kidneys irrespective of histologic changes (LRTx). In this report, we explored the reasons behind this guarded outcome with a special focus on the role of alloimmunity. We initially assessed gene expression in 39 time-zero allograft biopsies using the Nanostring 770 genes PanCancer Immune Profiling Panel. Subsequently, we studied 696 consecutive adult kidney allograft recipients that were grouped according to allograft type and histology at time-zero biopsy [DRTx/suboptimal histology (n = 194), DRTx/optimal histology (n = 166), and LRTx (n = 336)]. Part-1: Several immune pathways were upregulated in time-zero biopsies from DRTx/suboptimal histology (n = 11) compared to LRTx (n = 17) but not to DRTx/optimal histology (n = 11). Part-2: Amongst the three groups of recipients, DRTx/suboptimal histology had the highest incidence of acute rejection episodes, most of which occurred during the first year after transplantation (early rejection). This increase was mainly attributed to T cell mediated rejection, while the incidence of antibody-mediated rejection was similar amongst the three groups. Importantly, early acute T cell mediated rejection was a strong independent predictor for allograft failure in DRTx/suboptimal histology (adjusted HR: 2.13, P = 0.005) but not in DRTx/optimal histology nor in LRTx. Our data highlight an increased baseline immunogenicity in DRTx/suboptimal histology compared to LRTx but not to DRTx/optimal histology. However, our results suggest that donor chronic histologic changes in DRTx may help transfer such increased baseline immunogenicity into clinically relevant acute rejection episodes that have detrimental effects on allograft survival. These findings may provide a rationale for enhanced immunosuppression in recipients of DRTx with baseline chronic histologic changes to minimize subsequent acute rejection and to prolong allograft survival.

Masson Trichrome and Sulfated Alcian Blue Stains Distinguish Light Chain Deposition Disease From Amyloidosis in the Lung.

Light chain deposition disease, characterized by nonamyloidogenic deposits of immunoglobulin light chains, is rare in the lung and possibly underdiagnosed due to low clinical suspicion and lack of readily accessible tests. We encountered a case of pulmonary light chain deposition disease (PLCDD) in which light chain deposits appeared crimson red with a Masson trichrome (MT) stain and salmon pink with a sulfated Alcian blue (SAB) stain. This prompted us to characterize a series of PLCDD cases and assess the utility of MT and SAB stains to distinguish them from amyloidosis. From the pathology archives of 2 institutions spanning 10 years, we identified 11 cases of PLCDD, including 7 diagnosed as such and 4 determined retrospectively. The deposits in all cases of PLCDD stained crimson red with MT and salmon pink with SAB, while the cases of pulmonary amyloid (n=10) stained blue-gray and blue-green, respectively. The immunoglobulin light chain nature of the deposits was confirmed in 10 of 11 cases by either immunofluorescence microscopy (n=5) or mass spectrometry (n=5). Transmission electron microscopy revealed osmiophilic, electron-dense deposits in all cases analyzed (n=3). An extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue type was diagnosed in 10 cases and 1 represented a plasma cell neoplasm. Our study highlights the importance of considering PLCDD in the differential diagnosis of amyloid-like deposits in the lung and the value of performing MT and SAB stains to distinguish between PLCDD and amyloidosis.

Impact of Deceased Donor Kidney Procurement Biopsy Technique on Histologic Accuracy.

INTRODUCTION: The factors that influence deceased donor kidney procurement biopsy reliability are not well established. We examined the impact of biopsy technique and pathologist training on procurement biopsy accuracy. METHODS: We retrospectively identified all deceased donor kidney-only transplants at our center from 2006 to 2016 with both procurement and reperfusion biopsies performed and information available on procurement biopsy technique and pathologist (n = 392). Biopsies were scored using a previously validated system, classifying "suboptimal" histology as the presence of at least 1 of the following: glomerulosclerosis ≥11%, moderate/severe interstitial fibrosis/tubular atrophy, or moderate/severe vascular disease. We calculated relative risk ratios (RRR) to determine the influence of technique (core vs. wedge) and pathologist (renal vs. nonrenal) on concordance between procurement and reperfusion biopsy histologic classification. RESULTS: A total of 171 (44%) procurement biopsies used wedge technique, and 221 (56%) used core technique. Results of only 36 biopsies (9%) were interpreted by renal pathologists. Correlation between procurement and reperfusion glomerulosclerosis was poor for both wedge (r 2 = 0.11) and core (r 2 = 0.14) biopsies. Overall, 34% of kidneys had discordant classification on procurement versus reperfusion biopsy. Neither biopsy technique nor pathologist training was associated with concordance between procurement and reperfusion histology, but a larger number of sampled glomeruli was associated with a higher likelihood of concordance (adjusted RRR = 1.12 per 10 glomeruli, 95% confidence interval = 1.04-1.22). CONCLUSIONS: Biopsy technique and pathologist training were not associated with procurement biopsy histologic accuracy in this retrospective study. Prospective trials are needed to determine how to optimize procurement biopsy practices.

Combination checkpoint blockade for metastatic cutaneous malignancies in kidney transplant recipients.

BACKGROUND: Immune checkpoint blockade has emerged as a highly effective treatment for patients with metastatic melanoma and cutaneous squamous cell carcinoma. Nivolumab blocks the interactions between programmed cell death protein 1 and programmed death ligand 1 allowing for activation of a latent immune response against the malignancy. Ipilimumab binds to and blocks cytotoxic T-lymphocyte-associated protein 4, alleviating the negative regulation of T-cell activation that is mediated by that checkpoint. Combination therapy with nivolumab and ipilimumab is associated with longer overall survival at 5 years compared with nivolumab monotherapy. Solid organ transplant recipients have a significantly higher risk of malignancies compared with the general population. There is limited data surrounding the efficacy of combination immunotherapy in solid organ transplant recipients, as these patients were excluded from seminal trials due to risk of organ rejection. CASE PRESENTATIONS: Here we present four cases of combination immunotherapy in kidney transplant recipients. Three patients had metastatic melanoma, and one patient had metastatic cutaneous squamous cell carcinoma. Two patients had radiographic responses from immunotherapy, one patient had stable disease, and one patient had disease progression. Only one patient had biopsy-proven rejection. At last follow-up, three patients had functioning grafts, though one required hemodialysis after treatment, and one patient succumbed to disease, but graft function remained intact throughout her course. CONCLUSIONS: These cases describe the use of ipilimumab and nivolumab combination immunotherapy for cutaneous malignancies in kidney transplant recipients. They highlight the potential to preserve kidney graft function while effectively treating the disease. TRIAL REGISTRATION NUMBER: NCT03816332.

Large and Uneven Bites in End-to-End Anastomosis of the Rat Femoral Artery.

BACKGROUND: Successful microvascular anastomosis depends on sutures that adequately oppose both cut vessel edges. Trainees tend to take oversized or uneven bite. To improve early microsurgical skill acquisition using the rat, this study tests the belief that such bites compromise early patency by applying exaggerated bites to end-to-end arterial anastomoses. METHODS: Twelve Sprague-Dawley rats were randomly assigned to one of the four bite techniques to be applied to both femoral arteries (mean diameter, 0.8 mm). Large (L) and standard (S) bites measured 1.0 and 0.2 mm from the edge, respectively. Eight simple interrupted anastomoses were performed per bite technique, each labeled according to every proximal end bite size, followed by every distal end bite size: LL, LS, SL, and SS. Anastomosis time and blood flow rates were recorded and analyzed statistically. After sacrifice 5 days postoperation, anastomosis sections of each technique were examined histologically. RESULTS: All 24 anastomoses (100%) maintained patency for 5 days. There was no statistical difference between all postoperative blood flow measurements at any given time. Anastomosis times using LL, LS, SL, and SS bite techniques were 41.6, 33.2, 34.8, and 25.5 minutes, respectively. Anastomosis time for the traditional bite technique (SS) was significantly shorter than all other bite techniques (p < 0.05). Histological examination of the harvested segments from each group revealed similar pathophysiological features. CONCLUSION: Oversized bites (1 mm), placed symmetrically and asymmetrically across the anastomosis, do not affect early patency in the rat femoral artery. A reduced reliance on conventional guidelines for suture bites appears acceptable during microarterial anastomoses if the goal is vessel patency. However, we believe clinical competence involves the ability to place small, even bites consistently and uniformly. During microsurgical training, the occasional large bite need not be replaced; however, the trainee should be encouraged to take standard bites.

Reproducibility of Deceased Donor Kidney Procurement Biopsies.

BACKGROUND AND OBJECTIVES: Unfavorable histology on procurement biopsies is the most common reason for deceased donor kidney discard. We sought to assess the reproducibility of procurement biopsy findings. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We compiled a continuous cohort of deceased donor kidneys transplanted at our institution from 1/1/2006 to 12/31/2016 that had at least one procurement biopsy performed, and excluded cases with missing biopsy reports and those used in multiorgan transplants. Suboptimal histology was defined as the presence of advanced sclerosis in greater than or equal to one biopsy compartment (glomeruli, tubules/interstitium, vessels). We calculated κ coefficients to assess agreement in optimal versus suboptimal classification between sequential biopsy reports for kidneys that underwent multiple procurement biopsies and used time-to-event analysis to evaluate the association between first versus second biopsies and patient and allograft survival. RESULTS: Of the 1011 kidneys included in our cohort, 606 (60%) had multiple procurement biopsies; 98% had first biopsy performed at another organ procurement organization and their second biopsy performed locally. Categorical agreement was highest for vascular disease (κ=0.17) followed by interstitial fibrosis and tubular atrophy (κ=0.12) and glomerulosclerosis (κ=0.12). Overall histologic agreement (optimal versus suboptimal) was κ=0.15. First biopsy histology had no association with allograft survival in unadjusted or adjusted analyses. However, second biopsy optimal histology was associated with a higher probability of death-censored allograft survival, even after adjusting for donor and recipient factors (adjusted hazard ratio, 0.50; 95% confidence interval, 0.34 to 0.75; P=0.001). CONCLUSIONS: Deceased donor kidneys that underwent multiple procurement biopsies often displayed substantial differences in histologic categorization in sequential biopsies, and there was no association between first biopsy findings and post-transplant outcomes.

Methimazole-Induced Pauci-Immune Glomerulonephritis and Anti-Phospholipid Syndrome: An Important Association to Be Aware of.

While methimazole (MMI) is the first line treatment for hyperthyroidism, this medication is not devoid of adverse effects. In this article, we present a 70-year-old male who admitted the hospital with right lower extremity pain and rash. The patient was recently treated with MMI for hyperthyroidism. Imaging studies revealed bilateral renal and splenic infarcts along with thrombosis of popliteal artery. Laboratory data revealed hematuria and proteinuria with positive (MPO), anti-proteinase-3 (PR3) and anti-cardiolipin IgG antibodies. Renal biopsy revealed pauci-immune glomerulonephritis and features with anti-phospholipid antibody syndrome (APS). MMI was discontinued and the patient was treated successfully with steroid therapy and anti-coagulation with resolution of proteinuria, hematuria and normalization of laboratory parameters. While MMI-induced pauci-immune glomerulonephritis has been previously reported, its association with APS has never been described before. Our case demonstrates that this rare diagnosis can be treated by early withdrawal of MMI and initiation of steroids along with anticoagulation.

A molecular signature for delayed graft function.

Chronic kidney disease and associated comorbidities (diabetes, cardiovascular diseases) manifest with an accelerated ageing phenotype, leading ultimately to organ failure and renal replacement therapy. This process can be modulated by epigenetic and environmental factors which promote loss of physiological function and resilience to stress earlier, linking biological age with adverse outcomes post-transplantation including delayed graft function (DGF). The molecular features underpinning this have yet to be fully elucidated. We have determined a molecular signature for loss of resilience and impaired physiological function, via a synchronous genome, transcriptome and proteome snapshot, using human renal allografts as a source of healthy tissue as an in vivo model of ageing in humans. This comprises 42 specific transcripts, related through IFNγ signalling, which in allografts displaying clinically impaired physiological function (DGF) exhibited a greater magnitude of change in transcriptional amplitude and elevated expression of noncoding RNAs and pseudogenes, consistent with increased allostatic load. This was accompanied by increased DNA methylation within the promoter and intragenic regions of the DGF panel in preperfusion allografts with immediate graft function. Pathway analysis indicated that an inability to sufficiently resolve inflammatory responses was enabled by decreased resilience to stress and resulted in impaired physiological function in biologically older allografts. Cross-comparison with publically available data sets for renal pathologies identified significant transcriptional commonality for over 20 DGF transcripts. Our data are clinically relevant and important, as they provide a clear molecular signature for the burden of "wear and tear" within the kidney and thus age-related physiological capability and resilience.

Bidirectional intragraft alloreactivity drives the repopulation of human intestinal allografts and correlates with clinical outcome.

A paradigm in transplantation states that graft-infiltrating T cells are largely non-alloreactive "bystander" cells. However, the origin and specificity of allograft T cells over time has not been investigated in detail in animals or humans. Here, we use polychromatic flow cytometry and high throughput TCR sequencing of serial biopsies to show that gut-resident T cell turnover kinetics in human intestinal allografts are correlated with the balance between intra-graft host-vs-graft (HvG) and graft-vs-host (GvH) reactivities and with clinical outcomes. In the absence of rejection, donor T cells were enriched for GvH-reactive clones that persisted long-term in the graft. Early expansion of GvH clones in the graft correlated with rapid replacement of donor APCs by the recipient. Rejection was associated with transient infiltration by blood-like recipient CD28+ NKG2DHi CD8+ alpha beta T cells, marked predominance of HvG clones, and accelerated T cell turnover in the graft. Ultimately, these recipient T cells acquired a steady state tissue-resident phenotype, but regained CD28 expression during rejections. Increased ratios of GvH to HvG clones were seen in non-rejectors, potentially mitigating the constant threat of rejection posed by HvG clones persisting within the tissue-resident graft T cell population.

FNA, core biopsy, or both for the diagnosis of lung carcinoma: Obtaining sufficient tissue for a specific diagnosis and molecular testing.

BACKGROUND: Increasingly, minimally invasive procedures are performed to assess lung lesions and stage lung carcinomas. In cases of advanced-stage lung cancer, the biopsy may provide the only diagnostic tissue. The aim of this study was to determine which method-fine-needle aspiration (FNA), core biopsy (CBx), or both (B)--is optimal for providing sufficient tissue for rendering a specific diagnosis and pursuing molecular studies for guiding tumor-specific treatment. METHODS: A search was performed for computed tomography-guided lung FNA, CBx, or B cases with rapid onsite evaluation. Carcinomas were assessed for the adequacy to render a specific diagnosis; this was defined as enough refinement to subtype a primary carcinoma or to assess a metastatic origin morphologically and/or immunohistochemically. In cases of primary lung adenocarcinoma, the capability of each modality to yield sufficient tissue for molecular studies (epidermal growth factor receptor, KRAS, or anaplastic lymphoma kinase) was also assessed. RESULTS: There were 210 cases, and 134 represented neoplasms, including 115 carcinomas. For carcinomas, a specific diagnosis was reached in 89% of FNA cases (33 of 37), 98% of CBx cases (43 of 44), and 100% of B cases (34 of 34). For primary lung adenocarcinomas, adequate tissue remained to perform molecular studies in 94% of FNA cases (16 of 17), 100% of CBx cases (19 of 19), and 86% of B cases (19 of 22). No statistical difference was found among the modalities for either reaching a specific diagnosis (p = .07, Fisher exact test) or providing sufficient tissue for molecular studies (p = .30, Fisher exact test). CONCLUSIONS: The results suggest that FNA, CBx, and B are comparable for arriving at a specific diagnosis and having sufficient tissue for molecular studies: they specifically attained the diagnostic and prognostic goals of minimally invasive procedures for lung carcinoma.

Monoclonal IgG1κ anti-glomerular basement membrane disease: a case report.

We report a case of anti-glomerular basement membrane (anti-GBM) nephritis with indolent course, monoclonal IgG1κ (immunoglobulin G, subclass 1, κ light chain) linear staining of the GBM, and multifocal GBM breaks but without crescents or detectable serum anti-GBM antibody in a patient followed over 9 years. Atypically, anti-GBM nephritis follows an indolent course. A very small fraction of patients with anti-GBM nephritis lack detectable circulating anti-GBM antibodies, and rare reports of monoclonal anti-GBM nephritis exist. We report what is to our knowledge the first case manifesting all 3 of these rare variations. Our patient initially presented with asymptomatic decreased kidney function following an upper respiratory tract infection. He was found to have microhematuria and subnephrotic proteinuria with mild diffuse endocapillary proliferative and exudative glomerulonephritis with linear IgG1κ staining of the GBM. He was treated with an induction regimen of intravenous cyclophosphamide and corticosteroids followed by maintenance monotherapy with mycophenolic acid. Nine years later, repeat kidney biopsy for worsening kidney function after an upper respiratory tract infection showed persistent monoclonal staining of the GBM and acute glomerulonephritis with increased chronicity, including a single fibrocellular crescent. Despite extensive clinical investigations spanning nearly a decade, no circulating anti-GBM antibody or monoclonal protein has been detected. In this case report, we explore the unique features of this monoclonal IgG1κ-associated anti-GBM nephritis.

Granulomatous inflammation and organizing pneumonia: Role of computed tomography-guided lung fine needle aspirations, touch preparations and core biopsies in the evaluation of common non-neoplastic diagnoses.

BACKGROUND: Fine-needle aspirations (FNAs) and core biopsies (CBs), with or without touch preparations (TPs), are performed to characterize pulmonary lesions. Although a positive (P) or suspicious report is sufficient for further management, the significance of unsatisfactory (U), negative (N) and atypical (A) cytological diagnoses remains uncertain. The aims of the study were to correlate U, N and A cytological diagnoses with histological and/or clinical/radiological follow-up and evaluate the utility of FNAs, TPs and CBs. MATERIALS AND METHODS: We performed a retrospective search and examined 30 consecutive computed tomography-guided transthoracic U, N and A lung FNAs (n = 23) and TPs (n = 7) with surgical pathology (SP) (n = 17) and/or clinical/radiological follow-up (n = 13) and compared them to 10 SP-confirmed P FNAs, which served as controls. RESULTS: The 30 FNAs and TPs were from 29 patients. All 6 U specimens were scantly cellular. Granulomas, the most common specific benign cytological diagnosis, were evident in 8 (of 13) and 7 (of 11) N and A cytology cases, respectively. Histology corroborated the presence of granulomas identified on cytology. Organizing pneumonia was the second leading benign specific diagnosis (5/17), but it was rendered on histology (n = 5) and not FNAs or TPs. Evaluation of the A cases revealed that type II pneumocytes were the source of "atypical", diagnoses often associated with granulomas or organizing pneumonia and lacked 3-D clusters evident in all P cases. DISCUSSION: U, N and A FNAs and TPs lacked 3-D clusters seen in carcinomas and were negative on follow-up. Granulomas and organizing pneumonia were the most common specific benign diagnoses, but the latter was recognized on histology only. In the absence of a definitive FNA result at the time of on-site assessment, a CB with a TP containing type II pneumocytes increases the likelihood of a specific benign diagnosis.

High-frequency alloreactive T cells augment effector function of low-frequency CD8+ T-cell responses under CD28/CD154 blockade.

BACKGROUND: Blockade of costimulatory molecules is a potent method of inducing long-term graft survival. We have previously addressed the issue of donor-reactive T-cell precursor frequency on relative costimulation dependence and found that the presence of a high precursor frequency of donor-reactive CD8 T cells resulted in costimulation blockade-resistant graft rejection, whereas the presence of a low-frequency donor-reactive population did not. To address the mechanisms by which high-frequency T cells obviated the requirement for costimulation, we asked whether a low-frequency population responding concomitantly with a high-frequency response also demonstrated costimulation independence. METHODS: A model system was established in which B6 mice containing a low frequency of anti-membrane bound chicken ovalbumin (mOVA) responders and a high frequency of anti-BALB/c responders received a skin graft from B6.mOVAxBALB/c F1 donors in the presence or absence of cytotoxic T-lymphocyte antigen-4 Ig/anti-CD154 costimulatory blockade. RESULTS: The results revealed that in the presence of costimulation blockade, high-frequency anti-BALB/c T cells augmented the effector activity of low-frequency anti-mOVA T cells, but it did not enhance the accumulation of anti-mOVA T cells capable of mediating graft rejection. CONCLUSIONS: These results demonstrate that both antigen-specific and antigen-independent factors contribute to the relative costimulation independence of high-frequency T-cell responses.

IFN-gamma dictates allograft fate via opposing effects on the graft and on recipient CD8 T cell responses.

CD8 T cells are necessary for costimulation blockade-resistant rejection. However, the mechanism by which CD8 T cells mediate rejection in the absence of major costimulatory signals is poorly understood. IFN-gamma promotes CD8 T cell-mediated immune responses, but IFN-gamma-deficient mice show early graft loss despite costimulation blockade. In contrast, we found that IFN-gamma receptor knockout mice show dramatically prolonged graft survival under costimulation blockade. To investigate this paradox, we addressed the effects of IFN-gamma on T cell alloresponses in vivo independent of the effects of IFN-gamma on graft survival. We identified a donor-specific CD8 T cell breakthrough response temporally correlated with costimulation blockade-resistant rejection. Neither IFN-gamma receptor knockout recipients nor IFN-gamma-deficient recipients showed a CD8 breakthrough response. Graft death on IFN-gamma-deficient recipients despite costimulation blockade could be explained by the lack of IFN-gamma available to act on the graft. Indeed, the presence of IFN-gamma was necessary for graft survival on IFN-gamma receptor knockout recipients, as either IFN-gamma neutralization or the lack of the IFN-gamma receptor on the graft precipitated early graft loss. Thus, IFN-gamma is required both for the recipient to mount a donor-specific CD8 T cell response under costimulation blockade as well as for the graft to survive after allotransplantation.