Tissue-resident memory T (T_RM) cells are characterized by their surface expression of CD69 and can be subdivided in CD103+ and CD103− T_RM cells. The origin and functional characteristics of T_RM cells in the renal allograft are largely unknown. To determine these features we studied T_RM cells in transplant nephrectomies. T_RM cells with a CD103+ and CD103− phenotype were present in all samples (n = 13) and were mainly CD8+ T cells. Of note, donor-derived T_RM cells were only detectable in renal allografts that failed in the first month after transplantation. Grafts, which failed later, mainly contained recipient derived T_RM cells. The gene expression profiles of the recipient derived CD8+ T_RM cells were studied in more detail and showed a previously described signature of tissue residence within both CD103+ and CD103− T_RM cells. All CD8+ T_RM cells had strong effector abilities through the production of IFNγ and TNFα, and harboured high levels of intracellular granzyme B and low levels of perforin. In conclusion, our results demonstrate that donor and recipient T_RM cells reside in the rejected renal allograft. Over time, the donor-derived T_RM cells are replaced by recipient T_RM cells which have features that enables these cells to aggressively respond to the allograft.

Interaction between T follicular helper (Tfh) cells and B cells is complex and involves various pathways, including the production of IL-21 by the Tfh cells. Secretion of IL-21 results in B cell differentiation towards immunoglobulin producing plasmablasts. In patients after kidney transplantation, the formation of alloantibodies produced by donor antigen activated B cells are a major cause of organ failure. In this allogeneic response, the role of IL-21 producing Tfh cells that regulate B cell differentiation is unknown. Here, we tested in an alloantigen driven setting whether Tfh cell help signals control B cell differentiation with its dependency on IL-21. Pre transplantation patient PBMCs were FACS sorted into pure CD4posCXCR5pos Tfh cells and CD19posCD27pos memory B cells and stimulated with donor antigen in the presence or absence of an IL-21R antagonist (αIL-21R). Donor antigen stimulation initiated expression of the activation markers ICOS and PD-1 on Tfh cells, and a shift towards a mixed Tfh2 and Tfh17 phenotype. The memory B cells underwent class switch recombination (CSR) and differentiated towards IgM and IgG producing plasmablasts. In the presence of αIL-21R, a dose dependent inhibition of STAT3 phosphorylation was measured in both T and B cells. Blockade of the IL-21R did not have an effect on PD-1 and ICOS expression on Tfh cells but significantly inhibited B cell differentiation. The proportion of plasmablasts decreased by 78% in the presence of αIL-21R. Moreover, secreted IgM and IgG2 levels were significantly lower in the presence of αIL-21R. In conclusion, our results demonstrate that IL-21 produced by alloantigen activated Tfh cells controls B cell differentiation towards antibody producing plasmablasts. The IL-21R might therefore be a useful target in organ transplantation to prevent antigen driven immune responses leading to graft failure.

Over the past decade antibody-mediated (humoral) rejection has been recognized as a common cause of graft dysfunction after organ transplantation and an important determinant for graft loss. In humoral alloimmunity, T follicular helper (Tfh) cells play a crucial role, because they help naïve B cells to differentiate into memory B cells and alloantibody-producing plasma cells within germinal centers. In this way, they contribute to the induction of donor-specific antibodies, which are responsible for the humoral immune response to the allograft. In this article we provide an overview of the current knowledge on the effects of immunosuppressive therapies on Tfh cell development and function, and discuss possible new approaches to influence the activity of Tfh cells. In addition, we discuss the potential use of Tfh cells as a pharmacodynamic biomarker to improve alloimmune risk stratification and tailoring of immunosuppression in order to individualize therapy after transplantation.

We hypothesize that T cells such as interleukin (IL)-21⁺B cell lymphoma 6 (BCL6)⁺ T follicular helper cells can regulate B cell-mediated immunity within the allograft during acute T cell-mediated rejection; this process may feed chronic allograft rejection in the long term. To investigate this mechanism, we determined the presence and activation status of organized T and B cells in so-called ectopic lymphoid structures (ELSs) in different types of acute renal allograft rejection. Biopsies showing the following primary diagnosis were included: acute/active antibody-mediated rejection, C4d⁺ (a/aABMR), acute T cell-mediated rejection grade I (aTCMRI) and acute T cell-mediated rejection grade II (aTCMRII). Paraffin sections were stained for T cells (CD3 and CD4), B cells (CD20), follicular dendritic cells (FDCs, CD23), activated B cells (CD79A), immunoglobulin (Ig)D, cell proliferation (Ki67) and double immunofluorescent stainings for IL-21 and BCL6 were performed. Infiltrates of T cells were detected in all biopsies. In aTCMRI, B cells formed aggregates surrounded by T cells. In these aggregates, FDCs, IgD and Ki67 were detected, suggesting the presence of ELSs. In contrast, a/aABMR and aTCMRII showed diffuse infiltrates of T and B cells but no FDCs and IgD. IL-21 was present in all biopsies. However, co-localization with BCL6 was observed mainly in aTCMRI biopsies. In conclusion, ELSs with an activated phenotype are found predominantly in aTCMRI where T cells co-localize with B cells. These findings suggest a direct pathway of B cell alloactivation at the graft site during T cell mediated rejection.

Background In solid organ transplant (SOT) recipients, transplant rejection during immune checkpoint inhibitor (ICI) treatment for cancer is a clinical problem. Donor-derived cell-free DNA (dd-cfDNA) can be detected in blood and is a sensitive biomarker for diagnosis of acute rejection in SOT recipients. To our best knowledge, this is the first case report of a kidney transplant recipient with advanced cancer treated with ICI who was monitored with dd-cfDNA. Case presentation A 72-year old female with a long-standing renal transplant was diagnosed with advanced melanoma in 2018 and was treated with the anti-PD1 antibody nivolumab. Within 12 days after the first administration of nivolumab, dd-cfDNA ratio increased to 23%, suggesting allograft rejection. Her kidney transplant function deteriorated and acute rejection was confirmed by renal transplant biopsy. As the rejection could not be controlled despite immunosuppressive treatment, a transplant nephrectomy was necessary and haemodialysis was started. Immunological analysis of the renal explant showed infiltration of alloreactive, nivolumab-saturated, PD1+ cytotoxic T cells. After transplant nephrectomy, she experienced nivolumab-related toxicity and rapid disease progression. Conclusion Clinicians prescribing ICIs should be aware that SOT recipients are at risk of transplant rejection as a result of T cell activation. Dd-cfDNA is a sensitive biomarker and should be further studied for early detection of transplant rejection. Immunological analysis of the kidney explant showed marked graft infiltration with alloreactive PD-1⁺ cytotoxic T cells that were saturated with nivolumab.

Introduction: C‐peptide is an important marker to assess residual insulin production in individuals with type 1 diabetes (T1D). The accuracy and detection limits of C‐peptide assays are important to detect C‐peptide microsecretion and to reliably observe changes over time in these people. We compared and verified two commercially available assays able to measure C‐peptide in the picomolar range.Methods: The ultrasensitive Mercodia enzyme‐linked immunosorbent C‐peptide assay (ELISA) was compared with the Beckman immunoradiometric assay (IRMA) for C‐peptide, assessing reproducibility (coefficient of variation [CV]), limit of blank (LoB), limit of detection (LoD) and limit of quantitation (LoQ).Results: For both assays within‐run and between‐run variation were high at the low (around the detection limit) C‐peptide concentration range, with CVs of around 40%. LoB values for the ultrasensitive ELISA and the IRMA were 1.3 and 0.16 pmol/L respectively. LoD values were 2.4 and 0.54 pmol/L respectively. LoQ values were 9.7 and 3.8 pmol/L respectively. Only the IRMA met the specifications claimed by the manufacturer.Conclusions: The IRMA provided the lowest threshold for quantification of serum C‐peptide. LoQ of commercially available assays should be established in‐house before applying them in research studies and clinical trials in which low C‐peptide levels have clinical or scientific relevance.