Integration of ζ-deficient CARs into the CD3ζ gene conveys potent cytotoxicity in T and NK cells.

ABSTRACT: Chimeric antigen receptor (CAR)-redirected immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in nonphysiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR expression and redirection of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3ζ-CD19-CAR-T cells exhibited comparable leukemia control to TCRα chain constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3ζ-CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3ζ gene editing enabled redirection of NK cells without impairing their canonical functions. Thus, CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes.

Preformed T cell alloimmunity and HLA eplet mismatch to guide immunosuppression minimization with tacrolimus monotherapy in kidney transplantation: Results of the CELLIMIN trial.

Personalizing immunosuppression is a major objective in transplantation. Transplant recipients are heterogeneous regarding their immunological memory and primary alloimmune susceptibility. This biomarker-guided trial investigated whether in low immunological-risk kidney transplants without pretransplant DSA and donor-specific T cells assessed by a standardized IFN-γ ELISPOT, low immunosuppression (LI) with tacrolimus monotherapy would be non-inferior regarding 6-month BPAR than tacrolimus-based standard of care (SOC). Due to low recruitment rates, the trial was terminated when 167 patients were enrolled. ELISPOT negatives (E-) were randomized to LI (n = 48) or SOC (n = 53), E+ received the same SOC. Six- and 12-month BPAR rates were higher among LI than SOC/E- (4/35 [13%] vs. 1/43 [2%], p = .15 and 12/48 [25%] vs. 6/53 [11.3%], p = .073, respectively). E+ patients showed similarly high BPAR rates than LI at 6 and 12 months (12/55 [22%] and 13/66 [20%], respectively). These differences were stronger in per-protocol analyses. Post-hoc analysis revealed that poor class-II eplet matching, especially DQ, discriminated E- patients, notably E-/LI, developing BPAR (4/28 [14%] low risk vs. 8/20 [40%] high risk, p = .043). Eplet mismatch also predicted anti-class-I (p = .05) and anti-DQ (p < .001) de novo DSA. Adverse events were similar, but E-/LI developed fewer viral infections, particularly polyoma-virus-associated nephropathy (p = .021). Preformed T cell alloreactivity and HLA eplet mismatch assessment may refine current baseline immune-risk stratification and guide immunosuppression decision-making in kidney transplantation.

Preformed donor-reactive T cells that persist after ABO desensitization predict severe T cell-mediated rejection after living donor kidney transplantation - a retrospective study.

Preformed donor-reactive T cells are relatively resistant to standard immunosuppression and account for an increased incidence of T cell-mediated rejection (TCMR) and inferior kidney allograft outcomes. We analyzed 150 living donor kidney transplant recipients (KTRs) of a first kidney allograft. Ninety-eight ABO-compatible (ABOc) and 52 ABO-incompatible (ABOi) KTRs were included. Samples were collected at 6 time points, before rituximab, before immunoadsorption and pretransplantation, at +1, +2, and +3 months posttransplantation, and donor-reactive T cells were measured by interferon-γ ELISPOT assay. Twenty of 98 ABOc (20%) and 12 of 52 ABOi KTRs (23%) showed positive pretransplant ELISPOT. Eight of 20 ABOc-KTRs (40%) with positive pretransplant ELISPOT showed TCMR, whereas 17 of 78 ABOc-KTRs (22%) with negative pretransplant ELISPOT did (P = 0.148). Seven of 12 ABOi KTRs (57%) with positive pretransplant ELISPOT showed TCMR, whereas only 3 of 40 ABOi KTRs (8%) with negative pretransplant ELISPOT did (P < 0.001). Interestingly, 6 of 7 ABOi KTRs with positive pretransplant ELISPOT that persists after ABO desensitization developed TCMR. Among 118 KTRs with negative pretransplant ELISPOT, 10 of 72 ABOc-KTRs (14%), but 0 of 46 ABOi KTRs, developed positive posttransplant ELISPOT (P = 0.006). Preformed donor-reactive T cells that persist despite ABO desensitization identify KTRs at highest risk of TCMR. Less de-novo donor-reactive T cells after ABO desensitization may account for less TCMR. Both, the use of rituximab and early initiation of calcineurin inhibitor-based maintenance immunosuppression may contribute to these findings.

Differential T cell response against BK virus regulatory and structural antigens: A viral dynamics modelling approach.

BK virus (BKV) associated nephropathy affects 1-10% of kidney transplant recipients, leading to graft failure in about 50% of cases. Immune responses against different BKV antigens have been shown to have a prognostic value for disease development. Data currently suggest that the structural antigens and regulatory antigens of BKV might each trigger a different mode of action of the immune response. To study the influence of different modes of action of the cellular immune response on BKV clearance dynamics, we have analysed the kinetics of BKV plasma load and anti-BKV T cell response (Elispot) in six patients with BKV associated nephropathy using ODE modelling. The results show that only a small number of hypotheses on the mode of action are compatible with the empirical data. The hypothesis with the highest empirical support is that structural antigens trigger blocking of virus production from infected cells, whereas regulatory antigens trigger an acceleration of death of infected cells. These differential modes of action could be important for our understanding of BKV resolution, as according to the hypothesis, only regulatory antigens would trigger a fast and continuous clearance of the viral load. Other hypotheses showed a lower degree of empirical support, but could potentially explain the clearing mechanisms of individual patients. Our results highlight the heterogeneity of the dynamics, including the delay between immune response against structural versus regulatory antigens, and its relevance for BKV clearance. Our modelling approach is the first that studies the process of BKV clearance by bringing together viral and immune kinetics and can provide a framework for personalised hypotheses generation on the interrelations between cellular immunity and viral dynamics.

Transplantectomy is associated with presensitization with donor-reactive T cells and graft failure after kidney retransplantation: a cohort study.

Background: The number of kidney transplant recipients (KTRs) being waitlisted for a subsequent transplantation has disproportionately increased to almost 25%. Evidence for the optimal management of the failed allograft, however, remains inconsistent. Methods: We studied 111 KTRs who underwent their second kidney transplantation from 1998 to 2015. In 51/111 KTRs (46%) the failed allograft was removed and in 60/111 (54%) the failed allograft was retained. KTRs with primary non-function and allograft loss <12 months of the first failed allograft were excluded from analysis. Samples were collected before transplantation and at 1 month posttransplantation and donor-reactive T cells were measured using an interferon-γ enzyme-linked immunosorbent spot assay. Results: KTRs with the previous allograft removed showed significantly higher rates of acute cellular rejection compared with KTRs with the previous allograft retained [27/51 KTRs (53%) versus 18/60 KTRs (30%); P = 0.019]. KTRs with the previous allograft removed showed significantly inferior death-censored allograft survival compared with KTRs with the previous allograft retained (P = 0.022). Here, KTRs with the previous allograft removed showed significantly higher donor-reactive T cells pretransplantation compared with KTRs with the previous allograft retained (P = 0.012). Interestingly, no differences were observed for the presence of panel reactive antibodies and for the development of de novo donor-specific antibodies. Conclusions: Our data suggest higher cellular presensitization among KTRs with the previous allograft removed, which is associated with higher rates of acute cellular rejection and inferior allograft survival. Immunological mechanisms that may account for these differences may include prolonged maintenance immunosuppression to save urine output in KTRs with the first kidney allograft retained and cellular presensitization after withdrawal of maintenance immunosuppression, which lead to allograft rejection and ultimately to allograft nephrectomy.

Diabetic kidney transplant recipients: Impaired infection control and increased alloreactivity.

BACKGROUND: Post-transplantation diabetes mellitus (PTDM) has been associated with inferior patient and allograft outcomes. However, previous studies did not identify differences in infection control and alloreactivity. METHODS: We studied 449 kidney transplant recipients (KTRs) between 2005 and 2013. Fifty (11.1%) KTRs were diagnosed with PTDM and 60 (13.4%) KTRs with pre-existing diabetes. Samples were collected pretransplantation, at +1, +2, +3 months post-transplantation. CMV specific and alloreactive T cells were quantified by interferon-γ Elispot assay. Lymphocyte subpopulations were quantified by flow cytometry. RESULTS: Upon multivariate analysis, age, obesity, and the use of tacrolimus increased the risk of PTDM (P<.05). KTRs with pre-existing diabetes/PTDM showed higher rates of sepsis (P<.01). Total CD3+ and CD4+ T cell counts were significantly lower in KTRs with PTDM/pre-existing diabetes post-transplantation (P<.05). No differences were observed for CMV-specific T cells between any group (P>.05). KTRs developing PTDM showed increased frequencies of alloreactive T-cells post-transplantation (P<.05). CONCLUSIONS: Our results suggest higher rates of infection in KTRs with pre-existing diabetes/PTDM that may be attributed to impaired overall immunity. Higher frequencies of alloreactive T cells contribute to inferior long-term outcomes. As acute rejection, but not pre-existing diabetes/PTDM, was associated with inferior allograft survival and function, maintaining adequate immunosuppression to prevent rejection seems important.

Sepsis after renal transplantation: Clinical, immunological, and microbiological risk factors.

BACKGROUND: As immunosuppressive therapy and allograft survival have improved, the increased incidence of sepsis has become a major hurdle of disease-free survival after renal transplantation. METHODS: We identified 112 of 957 kidney transplant recipients (KTRs) with sepsis. In all, 31 KTRs developed severe sepsis or septic shock, and 30 KTRs died from sepsis. KTRs without sepsis were used for comparison. CMV-specific and alloreactive T cells were measured using an interferon-γ Elispot assay. The extent of immunosuppression was quantified by lymphocyte subpopulations. RESULTS: Five-year patient survival was 70.3% with sepsis compared to 88.2% without (P<.001). Five-year estimated glomerular filtration rate was lower in KTRs developing sepsis (P<.001). Upon multivariate analysis, diabetes, lymphocyte-depleting induction, donor age, CMV replication, and acute rejection increased the risk of sepsis (P<.05). Age, diabetes, underweight/obesity, and pneumonia as site of infection were predictive factors of mortality (P<.05). Early-onset sepsis was associated with decreased CD3+ and CD4+ T cells pre-transplantation (P<.05). Impaired CMV-specific cellular immunity pre-transplantation was associated with CMV replication and early-onset sepsis (P<.05). High frequencies of alloreactive T cells were associated with acute rejection, lymphocyte-depleting rejection treatment, and early-onset sepsis (P<.05). CONCLUSION: KTRs developing sepsis show inferior patient survival and allograft function. Identified risk factors and differences in lymphocyte counts, CMV-specific immunity, and alloreactivity may prove useful to identify KTRs at increased risk.

Kidney transplant recipients after nonrenal solid organ transplantation show low alloreactivity but an increased risk of infection.

The number of kidney transplant recipients (KTRs) after nonrenal solid organ transplantation (SOT) has increased to almost 5%. Knowledge on patient and allograft outcomes, infections, and alloreactivity, however, remains scarce. We studied 40 KTRs after nonrenal SOT. Seven hundred and twenty primary KTRs and 119 repeat KTRs were used for comparison. Samples were collected pretransplantation, at +1, +2, and +3 months post-transplantation. Alloreactive and CMV-specific T cells were measured by interferon-γ ELISPOT assay. Patient survival in KTRs after SOT, primary and repeat KTRs was comparable. While death-censored allograft survival was comparable between KTRs after SOT and primary KTRs, KTRs after SOT showed superior 5-year death-censored allograft survival of 92.5% compared to 81.2% in repeat KTRs. Interestingly, KTRs after SOT show less preformed panel-reactive antibodies, frequencies of alloreactive T cells, and acute rejections compared to repeat KTRs. KTRs after SOT, however, show higher incidences of EBV viremia and PTLD, sepsis, and death from sepsis. Impaired CMV-specific cellular immunity was associated with more CMV replication compared to repeat KTRs. Our results suggest comparable patient and allograft outcomes in KTRs after SOT and primary KTRs. The observed low alloreactivity may contribute to excellent allograft outcomes. Caution should be taken in KTRs after SOT regarding infectious complications due to overimmunosuppression.

ABO desensitization affects cellular immunity and infection control after renal transplantation.

The impact of ABO desensitization on overall immunity, infectious control, and alloreactivity remains unknown. We compared 35 ABO-incompatible kidney transplant recipients (KTRs) to a control of 62 ABO compatible KTRs. Samples were collected before, at +1, +2, +3, +6, and +12 months post-transplantation. CMV-, BKV-specific, and alloreactive T cells were measured using an interferon-γ ELISPOT assay. The extent of immunosuppression was quantified by enumeration of lymphocyte subpopulations and cytokines. No differences were observed for 5-year allograft survival and function between both groups (P > 0.05). However, ABO-incompatible KTRs were more likely to develop CMV infection, BKV-associated nephropathy, and severe sepsis (P = 0.001). Interestingly, ABO-incompatible KTRs with poor HLA-match showed the highest rates of infections and inferior allograft function (P < 0.05). CD3+, CD4+ T-cell counts, interferon-γ and IL-10 levels were lower in ABO-incompatible KTRs early post-transplantation (P < 0.05). Likewise, ABO-incompatible KTRs showed impaired BKV- and CMV-specific T-cell immunity (P < 0.05). ABO-incompatible KTRs showed lower frequencies of alloreactive T cells (P < 0.05). Our data suggest T-cell depletion due to ABO desensitization, which may contribute to the increased risk of T-cell-dependent infections. Elimination of B cells serving as antigen-presenting cells, thereby causing impaired T-cell activation, plays a significant role in both impaired infection control and reduced alloreactive T-cell activation.

Inflammatory activation and recovering BKV-specific immunity correlate with self-limited BKV replication after renal transplantation.

As BKV-associated nephropathy has emerged as an important cause of allograft failure, it has been of major importance to find immune mechanisms suitable to identify kidney transplant recipients (KTRs) at increased risk of BKV replication. We monitored 29 KTRs with seven measurements during the first year post-transplantation. BKV-specific T cells directed to 5 BKV proteins were analyzed in an interferon-γ ELISPOT assay. BKV-specific antibodies were measured using an ELISA. The extent of immunosuppression and inflammatory activation were quantified by measures of immune function including lymphocyte subpopulations, IP-10, and adhesion molecule serum levels. All 5 BKV-specific T cells increased significantly from diagnosis to resolution of BKV replication (P<0.001). While antistructural T cells were significantly higher in KTRs with BKV replication (P<0.05), no differences were observed for antismall t- and large T-antigen-directed T cells (P>0.05). Interestingly, 65% of KTRs without BKV replication showed transient appearance of antismall t- and large T-antigen-directed T cells. Although no significant differences were observed for T-cell subpopulations and adhesion molecules, IP-10 levels increased significantly during BKV replication (P<0.05). Assessment of BKV-specific T cells identifies recovering BKV-specific immunity in KTRs with BKV replication and suggests their protective ability in KTRs without BKV replication. Increases in IP-10 levels stress the importance of infiltrating inflammatory leukocytes in the regulation of BKV replication and point to inflammatory activation in the pathogenesis of BKV replication.

Tacrolimus After rATG and Infliximab Induction Immunosuppression-RIMINI Trial.

BACKGROUND: Infliximab selectively targets recently activated effector cells and, as an induction agent, might enable the safe elimination of mycophenolate from maintenance immunosuppression in kidney transplantation. METHODS: This is a phase II international multicenter open-label single-arm confidence interval (CI)-based clinical trial of the BIO-DrIM EU consortium aimed at assessing the efficacy and safety of rabbit antithymocyte globulin and infliximab induction in kidney transplantation. Sixty-seven primary kidney transplant recipients at low risk (panel-reactive antibodies <20%, no donor-specific antibodies [DSA]) received rabbit antithymocyte globulin (2 × 1.5 mg/kg, postoperative days 0 and 1) and infliximab (5 mg/kg, postoperative day 2), followed by mycophenolate-free tacrolimus-based immunosuppression for 12 mo. The primary endpoint was efficacy failure, defined as a composite of acute rejection, graft loss, or poor graft function (estimated glomerular filtration rate <40 mL/min) at 12 mo and was based on the endpoint of the comparator study. Additionally, a historical propensity-matched control cohort was established. RESULTS: Primary endpoint occurred in 22 of 67 patients (32.84%), with upper bound of an exact 1-sided 95% CI of 43.47%, which met the predefined criteria (efficacy failure of <40% and upper-bound 95% CI of <50%) and was similar in the historical matched cohort. By 12 mo, 79.1% of patients remained on the study protocol. Lower rates of BK replication (6% versus 22.4%; P = 0.013) but higher rates of de novo DSAs (11.9% versus 1.5%; P = 0.039) were observed in the study cohort. CONCLUSIONS: A similar efficacy of the study immunosuppression regimen to the comparator study and the historical matched cohort was found. However, a higher de novo DSA emergence points to an increased risk of antibody-mediated rejection (NCT04114188).

Effector memory-type regulatory T cells display phenotypic and functional instability.

Regulatory T cells (Treg cells) hold promise for sustainable therapy of immune disorders. Recent advancements in chimeric antigen receptor development and genome editing aim to enhance the specificity and function of Treg cells. However, impurities and functional instability pose challenges for the development of safe gene-edited Treg cell products. Here, we examined different Treg cell subsets regarding their fate, epigenomic stability, transcriptomes, T cell receptor repertoires, and function ex vivo and after manufacturing. Each Treg cell subset displayed distinct features, including lineage stability, epigenomics, surface markers, T cell receptor diversity, and transcriptomics. Earlier-differentiated memory Treg cell populations, including a hitherto unidentified naïve-like memory Treg cell subset, outperformed late-differentiated effector memory-like Treg cells in regulatory function, proliferative capacity, and epigenomic stability. High yields of stable, functional Treg cell products could be achieved by depleting the small effector memory-like Treg cell subset before manufacturing. Considering Treg cell subset composition appears critical to maintain lineage stability in the final cell product.

Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells.

BACKGROUND: Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out or targeted transgene knock-in. However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells. RESULTS: Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted knock-in and Cas9-derived base editing technology for DSB free knock-outs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two knock-outs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduces translocations to 1.4% of edited cells. Small insertions and deletions at the base editing target sites indicate guide RNA exchange between the editors. This is overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR knock-in and a Cas9-derived base editor enables the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting TCR- and MHC-negative CAR T cells resist allogeneic T cell targeting in vitro. CONCLUSIONS: We outline a solution for non-viral CAR gene transfer and efficient gene silencing using different CRISPR enzymes for knock-in and base editing to prevent translocations. This single-step procedure may enable safer multiplex-edited cell products and demonstrates a path towards off-the-shelf CAR therapeutics.

CAR NK-92 cell-mediated depletion of residual TCR+ cells for ultrapure allogeneic TCR-deleted CAR T-cell products.

Graft-versus-host disease (GVHD) is a major risk of the administration of allogeneic chimeric antigen receptor (CAR)-redirected T cells to patients who are HLA unmatched. Gene editing can be used to disrupt potentially alloreactive T-cell receptors (TCRs) in CAR T cells and reduce the risk of GVHD. Despite the high knockout rates achieved with the optimized methods, a subsequent purification step is necessary to obtain a safe allogeneic product. To date, magnetic cell separation (MACS) has been the gold standard for purifying TCRα/ß- CAR T cells, but product purity can still be insufficient to prevent GVHD. We developed a novel and highly efficient approach to eliminate residual TCR/CD3+ T cells after TCRα constant (TRAC) gene editing by adding a genetically modified CD3-specific CAR NK-92 cell line during ex vivo expansion. Two consecutive cocultures with irradiated, short-lived, CAR NK-92 cells allowed for the production of TCR- CAR T cells with <0.01% TCR+ T cells, marking a 45-fold reduction of TCR+ cells compared with MACS purification. Through an NK-92 cell-mediated feeder effect and circumventing MACS-associated cell loss, our approach increased the total TCR- CAR T-cell yield approximately threefold while retaining cytotoxic activity and a favorable T-cell phenotype. Scaling in a semiclosed G-Rex bioreactor device provides a proof-of-principle for large-batch manufacturing, allowing for an improved cost-per-dose ratio. Overall, this cell-mediated purification method has the potential to advance the production process of safe off-the-shelf CAR T cells for clinical applications.

Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells.

Chimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3 ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3 ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3 ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3 ζ-CD19-CAR-T cells exhibited comparable leukemia control to T cell receptor alpha constant ( TRAC )-replaced and lentivirus-transduced CAR-T cells in vivo . Tuning of CD3 ζ-CAR-expression levels significantly improved the in vivo efficacy. Compared to TRAC -edited CAR-T cells, integration of a Her2-CAR into CD3 ζ conveyed similar in vitro tumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably, CD3 ζ gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus, CD3 ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes. Key points: Integration of ζ-deficient CARs into CD3 ζ gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf use CD3 ζ-editing platform allows CAR reprogramming of NK cells without affecting their canonical functions.

Tacrolimus CYP3A Single-Nucleotide Polymorphisms and Preformed T- and B-Cell Alloimmune Memory Improve Current Pretransplant Rejection-Risk Stratification in Kidney Transplantation.

Achieving fast immunosuppression blood exposure after kidney transplantation is key to abrogating both preformed and de novo anti-donor humoral and cellular alloresponses. However, while tacrolimus (TAC) is the cornerstone immunosuppressant inhibiting adaptive alloimmunity, its blood exposure is directly impacted by different single-nucleotide polymorphisms (SNPs) in CYP3A TAC-metabolizing enzymes. Here, we investigated how functional TAC-CYP3A genetic variants (CYP3A4*22/CYP3A5*3) influence the main baseline clinical and immunological risk factors of biopsy-proven acute rejection (BPAR) by means of preformed donor-specific antibodies (DSAs) and donor-specific alloreactive T cells (DSTs) in a large European cohort of 447 kidney transplants receiving TAC-based immunosuppression. A total of 70 (15.7%) patients developed BPAR. Preformed DSAs and DSTs were observed in 12 (2.7%) and 227 (50.8%) patients, respectively. According to the different CYP3A4*22 and CYP3A5*3 functional allele variants, we found 4 differential new clusters impacting fasting TAC exposure after transplantation; 7 (1.6%) were classified as high metabolizers 1 (HM1), 71 (15.9%) as HM2, 324 (72.5%) as intermediate (IM), and 45 (10.1%) as poor metabolizers (PM1). HM1/2 showed significantly lower TAC trough levels and higher dose requirements than IM and PM (p < 0.001) and more frequently showed TAC underexposure (<5 ng/ml). Multivariate Cox regression analyses revealed that CYP3A HM1 and IM pharmacogenetic phenotypes (hazard ratio (HR) 12.566, 95% CI 1.99-79.36, p = 0.007, and HR 4.532, 95% CI 1.10-18.60, p = 0.036, respectively), preformed DSTs (HR 3.482, 95% CI 1.99-6.08, p < 0.001), DSAs (HR 4.421, 95% CI 1.63-11.98, p = 0.003), and delayed graft function (DGF) (HR 2.023, 95% CI 1.22-3.36, p = 0.006) independently predicted BPAR. Notably, a significant interaction between T-cell depletion and TAC underexposure was observed, showing a reduction of the BPAR risk (HR 0.264, 95% CI 0.08-0.92, p = 0.037). Such variables except for DSAs displayed a higher predictive risk for the development of T cell-mediated rejection (TCMR). Refinement of pretransplant monitoring by incorporating TAC CYP3A SNPs with preformed DSAs as well as DSTs may improve current rejection-risk stratification and help induction treatment decision-making.

Pharmacological interventions enhance virus-free generation of TRAC-replaced CAR T cells.

Chimeric antigen receptor (CAR) redirected T cells are potent therapeutic options against hematological malignancies. The current dominant manufacturing approach for CAR T cells depends on retroviral transduction. With the advent of gene editing, insertion of a CD19-CAR into the T cell receptor (TCR) alpha constant (TRAC) locus using adeno-associated viruses for gene transfer was demonstrated, and these CD19-CAR T cells showed improved functionality over their retrovirally transduced counterparts. However, clinical-grade production of viruses is complex and associated with extensive costs. Here, we optimized a virus-free genome-editing method for efficient CAR insertion into the TRAC locus of primary human T cells via nuclease-assisted homology-directed repair (HDR) using CRISPR-Cas and double-stranded template DNA (dsDNA). We evaluated DNA-sensor inhibition and HDR enhancement as two pharmacological interventions to improve cell viability and relative CAR knockin rates, respectively. While the toxicity of transfected dsDNA was not fully prevented, the combination of both interventions significantly increased CAR knockin rates and CAR T cell yield. Resulting TRAC-replaced CD19-CAR T cells showed antigen-specific cytotoxicity and cytokine production in vitro and slowed leukemia progression in a xenograft mouse model. Amplicon sequencing did not reveal significant indel formation at potential off-target sites with or without exposure to DNA-repair-modulating small molecules. With TRAC-integrated CAR+ T cell frequencies exceeding 50%, this study opens new perspectives to exploit pharmacological interventions to improve non-viral gene editing in T cells.

Freezing Medium Containing 5% DMSO Enhances the Cell Viability and Recovery Rate After Cryopreservation of Regulatory T Cell Products ex vivo and in vivo.

Cell therapies have significant therapeutic potential in diverse fields including regenerative medicine, transplantation tolerance, and autoimmunity. Within these fields, regulatory T cells (Treg) have been deployed to ameliorate aberrant immune responses with great success. However, translation of the cryopreservation strategies employed for other cell therapy products, such as effector T cell therapies, to Treg therapies has been challenging. The lack of an optimized cryopreservation strategy for Treg products presents a substantial obstacle to their broader application, particularly as administration of fresh cells limits the window available for sterility and functional assessment. In this study, we aimed to develop an optimized cryopreservation strategy for our CD4+CD25+Foxp3+ Treg clinical product. We investigate the effect of synthetic or organic cryoprotectants including different concentrations of DMSO on Treg recovery, viability, phenotype, cytokine production, suppressive capacity, and in vivo survival following GMP-compliant manufacture. We additionally assess the effect of adding the extracellular cryoprotectant polyethylene glycol (PEG), or priming cellular expression of heat shock proteins as strategies to improve viability. We find that cryopreservation in serum-free freezing medium supplemented with 10% human serum albumin and 5% DMSO facilitates improved Treg recovery and functionality and supports a reduced DMSO concentration in Treg cryopreservation protocols. This strategy may be easily incorporated into clinical manufacture protocols for future studies.

The TreaT-Assay: A Novel Urine-Derived Donor Kidney Cell-Based Assay for Prediction of Kidney Transplantation Outcome.

Donor-reactive immunity plays a major role in rejection after kidney transplantation, but analysis of donor-reactive T-cells is not applied routinely. However, it has been shown that this could help to identify patients at risk of acute rejection. A major obstacle is the limited quantity or quality of the required allogenic stimulator cells, including a limited availability of donor-splenocytes or an insufficient HLA-matching with HLA-bank cells. To overcome these limitations, we developed a novel assay, termed the TreaT (Transplant reactive T-cells)-assay. We cultivated renal tubular epithelial cells from the urine of kidney transplant patients and used them as stimulators for donor-reactive T-cells, which we analyzed by flow cytometry. We could demonstrate that using the TreaT-assay the quantification and characterization of alloreactive T-cells is superior to other stimulators. In a pilot study, the number of pre-transplant alloreactive T-cells negatively correlated with the post-transplant eGFR. Frequencies of pre-transplant CD161+ alloreactive CD4+ T-cells and granzyme B producing alloreactive CD8+ T-cells were substantially higher in patients with early acute rejection compared to patients without complications. In conclusion, we established a novel assay for the assessment of donor-reactive memory T-cells based on kidney cells with the potential to predict early acute rejection and post-transplant eGFR.

CMV-Specific T Cell Monitoring Offers Superior Risk Stratification of CMV-Seronegative Kidney Transplant Recipients of a CMV-Seropositive Donor.

BACKGROUND: Detectable cytomegalovirus (CMV)-specific T cells in CMV-seronegative kidney transplant recipients (KTRs) have been attributed to an absence of circulating antibodies despite CMV sensitization. The diagnostic value of CMV-specific T cells, however, needs to be implemented in risk stratification for CMV replication. METHODS: Three hundred twenty-six KTRs were studied and classified with respect to CMV serostatus and presence of CMV-specific T cells. Samples were collected pretransplantation, at +1, +2, and +3 months posttransplantation. CMV-specific T cells directed to CMV-IE1 and CMV-pp65 were measured by interferon-γ Elispot assay. RESULTS: Nineteen (28%) of 67 D+R- KTRs showed pretransplant CMV-specific T cells. Although no differences were observed for CMV replication, KTRs with CMV-specific T cells presented with lower initial and peak CMV loads (P < 0.05). KTRs with decreasing/undetectable CMV-IE1-specific T cells pretransplantation and posttransplantation were at greatest risk of CMV replication. KTRs with stable/increasing CMV-IE1-specific T cells from pretransplantation to posttransplantation, however, showed low risk of CMV replication (P < 0.001). One hundred sixty-two (80%) of 203 R+ KTRs showed pretransplant CMV-specific T cells. Decreasing/undetectable CMV-IE1-specific T cells from pretransplantation and posttransplantation identified those R+ KTRs at increased risk of CMV replication (65/80 KTRs; 81%; P < 0.001). CONCLUSIONS: Despite CMV prophylaxis, D+R- KTRs are at greatest risk of CMV disease. Our data suggest that monitoring CMV-specific T cell kinetics from pretransplantation to posttransplantation, particularly directed to CMV-IE1, offers superior risk stratification compared with CMV serostatus alone.