In organ transplantation, infection and rejection are major causes of graft loss linked by the net state of immunosuppression. To diagnose and treat these conditions earlier, and to improve long-term patient outcomes, refined strategies for the monitoring of patients after graft transplantation are needed. Here, we show that a fast and inexpensive assay based on CRISPR-Cas13 accurately detects BK polyomavirus DNA and cytomegalovirus DNA from patient-derived blood and urine samples, as well as CXCL9 mRNA (a marker of graft rejection) at elevated levels in urine samples from patients experiencing acute renal-transplant rejection. The assay, which we adapted for lateral-flow readout, enables via simple visualization the post-transplantation monitoring of common opportunistic viral infections and of graft rejection, and should facilitate point-of-care posttransplantation monitoring.

BackgroundDeveloping a noninvasive clinical test to accurately diagnose kidney allograft rejection is critical to improve allograft outcomes. Urinary exosomes, tiny vesicles released into the urine that carry parent cells’ proteins and nucleic acids, reflect the biologic function of the parent cells within the kidney, including immune cells. Their stability in urine makes them a potentially powerful tool for liquid biopsy and a noninvasive diagnostic biomarker for kidney-transplant rejection.MethodsUsing 192 of 220 urine samples with matched biopsy samples from 175 patients who underwent a clinically indicated kidney-transplant biopsy, we isolated urinary exosomal mRNAs and developed rejection signatures on the basis of differential gene expression. We used crossvalidation to assess the performance of the signatures on multiple data subsets.ResultsAn exosomal mRNA signature discriminated between biopsy samples from patients with all-cause rejection and those with no rejection, yielding an area under the curve (AUC) of 0.93 (95% CI, 0.87 to 0.98), which is significantly better than the current standard of care (increase in eGFR AUC of 0.57; 95% CI, 0.49 to 0.65). The exosome-based signature’s negative predictive value was 93.3% and its positive predictive value was 86.2%. Using the same approach, we identified an additional gene signature that discriminated patients with T cell–mediated rejection from those with antibody-mediated rejection (with an AUC of 0.87; 95% CI, 0.76 to 0.97). This signature’s negative predictive value was 90.6% and its positive predictive value was 77.8%.ConclusionsOur findings show that mRNA signatures derived from urinary exosomes represent a powerful and noninvasive tool to screen for kidney allograft rejection. This finding has the potential to assist clinicians in therapeutic decision making.

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IntroductionStudies have shown reduced antiviral responses in kidney transplant recipients (KTRs) following SARS-CoV-2 mRNA vaccination, but data on post-vaccination alloimmune responses and antiviral responses against the Delta (B.1.617.2) variant are limited.Materials and methodsTo address this issue, we conducted a prospective, multi-center study of 58 adult KTRs receiving mRNA-BNT162b2 or mRNA-1273 vaccines. We used multiple complementary non-invasive biomarkers for rejection monitoring including serum creatinine, proteinuria, donor-derived cell-free DNA, peripheral blood gene expression profile (PBGEP), urinary CXCL9 mRNA and de novo donor-specific antibodies (DSA). Secondary outcomes included development of anti-viral immune responses against the wild-type and Delta variant of SARS-CoV-2.ResultsAt a median of 85 days, no KTRs developed de novo DSAs and only one patient developed acute rejection following recent conversion to belatacept, which was associated with increased creatinine and urinary CXCL9 levels. During follow-up, there were no significant changes in proteinuria, donor-derived cell-free DNA levels or PBGEP. 36% of KTRs in our cohort developed anti-wild-type spike antibodies, 75% and 55% of whom had neutralizing responses against wild-type and Delta variants respectively. A cellular response against wild-type S1, measured by interferon-γ-ELISpot assay, developed in 38% of KTRs. Cellular responses did not differ in KTRs with or without antibody responses.ConclusionsSARS-CoV-2 mRNA vaccination in KTRs did not elicit a significant alloimmune response. About half of KTRs who develop anti-wild-type spike antibodies after two mRNA vaccine doses have neutralizing responses against the Delta variant. There was no association between anti-viral humoral and cellular responses.

Programmed death-1 (PD-1) and its ligand PD-L1 are checkpoint molecules which regulate immune responses. Little is known about their functions in T cell migration and there are contradictory data about their roles in regulatory T cell (Treg) function. Here we show activated Tregs and CD4 effector T cells (Teffs) use PD-1/PD-L1 and CD80/PD-L1, respectively, to regulate transendothelial migration across lymphatic endothelial cells (LECs). Antibody blockade of Treg PD-1, Teff CD80 (the alternative ligand for PD-L1), or LEC PD-L1 impairs Treg or Teff migration in vitro and in vivo. PD-1/PD-L1 signals through PI3K/Akt and ERK to regulate zipper junctional VE-cadherin, and through NFκB-p65 to up-regulate VCAM-1 expression on LECs. CD80/PD-L1 signaling up-regulates VCAM-1 through ERK and NFκB-p65. PD-1 and CD80 blockade reduces tumor egress of PD-1high fragile Tregs and Teffs into draining lymph nodes, respectively, and promotes tumor regression. These data provide roles for PD-L1 in cell migration and immune regulation.

Respiratory syncytial virus (RSV) is a common cause of childhood lower respiratory tract infections. The recent failure of a vaccine candidate based on recombinant F protein underlines the urgent need to better understand the protective human memory immune response against RSV. Signal transducer and activator of transcription 3 (STAT3) protein is a transcription factor that promotes the maturation of the memory CD8 T cell response in cooperation with IL-10 and IL-21. However, the role of STAT3 in the memory CD8 T cell response during RSV infection remains to be elucidated. We found that in infants with bronchiolitis infected with RSV, the expression of STAT3 detected in nasal washes is reduced when compared to that in infants infected by other viruses. In vitro, RSV impairs STAT3 phosphorylation induced by IL-21 in purified human memory CD8 T cells. In addition, RSV decreases granzyme B production by memory CD8 T cells, reducing its cytotoxic activity against RSV-infected epithelial pulmonary cell lines. Together, these data indicate that RSV modulates the IL-21/STAT3 pathway in human memory CD8 T cells, and this could be a mechanism to be further explored to improve the memory response against the infection.

The programmed death 1/programmed death ligand 1 (PD-1/PD-L1) pathway is a potent inhibitory pathway involved in immune regulation and is a potential therapeutic target in transplantation. In this study, we show that overexpression of PD-1 on T cells (PD-1 Tg) promotes allograft tolerance in a fully MHC-mismatched cardiac transplant model when combined with costimulation blockade with CTLA-4–Ig. PD-1 overexpression on T cells also protected against chronic rejection in a single MHC II–mismatched cardiac transplant model, whereas the overexpression still allowed the generation of an effective immune response against an influenza A virus. Notably, Tregs from PD-1 Tg mice were required for tolerance induction and presented greater ICOS expression than those from WT mice. The survival benefit of PD-1 Tg recipients required ICOS signaling and donor PD-L1 expression. These results indicate that modulation of PD-1 expression, in combination with a costimulation blockade, is a promising therapeutic target to promote transplant tolerance.

Background: Available SARS-CoV-2 vaccines have reduced efficacy against the Omicron variant in immunocompetent individuals. Kidney transplant recipients (KTRs) have diminished antiviral responses to wild-type SARS-CoV-2 after vaccination, and data on antiviral responses to SARS-CoV-2 variants, including the Omicron variant, are limited. Methods: We conducted a prospective, multi-center cohort study of 51 adult KTRs who received three doses of BNT162b2 or mRNA-1273. Blood and urine samples were collected before and four weeks after the third vaccine dose. The primary outcome was anti-viral antibody responses against wild-type and variants of SARS-CoV-2. Secondary objectives included occurrence of breakthrough SARS-CoV-2 infection and non-invasive monitoring for rejection using serum creatinine, proteinuria, donor-derived cell-free DNA and donor-specific antibodies. Sera from pre-pandemic healthy controls and KTRs were used for comparison. Results: 67% of KTRs developed anti-wild-type spike antibodies after the third vaccine dose, similar to the Alpha (51%) and Beta (53%) variants, but higher than the Gamma (39%) and Delta (25%) variants. No KTRs had neutralizing responses to the Omicron variant before the third vaccine dose. After the third dose, fewer KTRs had neutralizing responses to the Omicron variant (12%) compared to wild-type (61%) and Delta (59%) variants. Three patients (6%) developed breakthrough SARS-CoV-2 infection at a median of 89 days. No KTRs developed allograft injury, de novo donor-specific antibodies or allograft rejection. Conclusion: In KTRs, a third dose of mRNA vaccines increases antibody responses against wild-type and variants of SARS-CoV-2, while neutralizing responses to the Omicron variant remain markedly reduced.