Impact of Circulating Lymphoma Cells on HLA Typing Outcomes in Patients with Diffuse Large B-Cell Lymphoma: A Case Report.

BACKGROUND: Recipient's high resolution HLA typing is required in allogeneic hematopoietic cell transplantation from unrelated donors, as well as for haploidentical family donors. For these purposes, Next-Generation Sequencing (NGS) methods are the gold standard. METHODS: We present a case of a patient with an incorrect HLA typing result caused by the population of circulating lymphoma cells. The first HLA examination was performed from peripheral blood (PB) using NGS in the active phase of diffuse large B-cell lymphoma with bone marrow involvement. RESULTS: Because of rare and inconclusive results, confirmed twice for the A* locus (A*02:32N), real-time polymerase chain reaction (RT-PCR)was performed. With RT-PCR method, we obtained more expected results according to the population allele frequency: in HLA-A locus (A*02:01) but also in DQB1 (DQB1*03:01, not as in NGS - DQB1*03:10). For the final verification, we used swab material and we obtained unambiguous NGS result with expected, frequent HLA-A*02:01 and DQB1*03:01 alleles corresponding to the RT-PCR result from PB. CONCLUSIONS: To conclude, we suspect that the discrepancies between NGS and RT-PCR results were caused by the presence of a significant amount of circulating lymphoma cells in the peripheral blood sample. Lymphomagenic mutations may involve the histocompatibility antigen coding region and affect HLA expressed on malignant cells. This finding may be relevant for the selection of test material in primary and confirmatory HLA testing in patients with active hematological malignancies because of the strong impact of incorrect HLA typing on the procedure of a donor selection.

Incidence and Role of Recipient-Specific Antibodies in Allogeneic Hematopoietic Cell Transplantation from Mismatched Related Donors.

High titer of donor-specific antibodies (DSAs) increases the risk of graft rejection after mismatched related hematopoietic cell transplantation (HCT). There are no data regarding the incidence of anti-HLA recipient-specific antibodies (RSAs) and their role after transplantation. Here we aimed to identify the incidence of RSAs in a mismatched related hematopoietic cell donor population and their possible impact on immune-mediated complications, such as acute graft-versus-host disease (aGVHD), and complications resulting from endothelial injury, such as transplantation-associated thrombotic microangiopathy (TA-TMA) and veno-occlusive disease (VOD). We prospectively analyzed the incidence of anti-HLA antibodies in 28 mismatched related pairs of recipients and their donors who underwent HCT at our center between 2020 and 2022. In positive samples screened for anti-HLA class I and/or II antibodies, the specificity of the HLA antibodies was analyzed. All recipients had a hematologic malignancy and received a myeloablative conditioning regimen and immunosuppression consisting of post-transplantation cyclophosphamide, tacrolimus, and mycophenolate mofetil. Patients were tested for TA-TMA and aGVHD development during routine post-transplantation visits up to 100 days post-transplantation. We used modified Jodele criteria for TA-TMA diagnosis, and based aGVHD grading on the MAGIC criteria. VOD was assessed using the European Society for Blood and Marrow Transplantation. Anti-HLA antibodies were detected in 12 donors (43%) and in 9 recipients (32%). There were no significant differences between donors and recipients according to age (median, 42 years [range, 17 to 69 years] versus 39 years [range, 8 to 68 years]), sex, or pregnancy history. No transfusion history was noted in the donor group (P < .05). RSA antibodies were present more often than DSAs and were detected in 9 out of 12 (75%) anti-HLA-positive donors and in only 2 out of 9 (22%) recipients, respectively (P < .05). During the follow-up, 11 patients (39%) developed aGVHD, including grade I-II in 9 (32%) and grade III-IV in 2 (7%). Twelve patients (43%) met the criteria for TA-TMA, and only 1 patient (3.5%) was diagnosed with VOD by day 100 post-HCT. RSAs were detected significantly more often in the TA-TMA group; among 12 patients diagnosed with TA-TMA, 7 (58%) had RSAs (P < .05). We did not find a correlation between RSAs and aGVHD. The patient with VOD did not have an RSA-positive donor. There was no difference in membrane attack complex (MAC) concentration in the RSA-positive group on day 30 and day 60 post-HCT; however, there was a trend toward higher MAC concentration in the RSA-positive group on day 100 (median, 912 ng/mL [range, 788 to 1120 ng/mL] versus 616 ng/mL [range, 352 to 1244 ng/mL]; P = .055). Patients with RSA suffered more often from platelet and red blood cell decreases or transfusion refractoriness, and increased lactate dehydrogenase activity was observed in all RSA-positive cases. The donor immune status and the presence of RSA may be associated with higher rates of TA-TMA in mismatched HCT recipients. Antibody-mediated complement activation might be an additional factor influencing TA-TMA occurrence.

Treatment of Patients After Lung Transplantation With Covid Infection During Long-Term Follow-Up.

BACKGROUND: Patients undergoing lung transplantation are routinely managed with lifelong immunosuppression, which is associated with a heightened risk for infections. This study delves into the therapeutic challenges and strategies for managing lung transplant recipients (LTRs) infected with COVID-19 during long-term follow-up. METHODS: The was a case series analysis, among which nonstandard therapies consisting of targeted antibody treatment, antiviral drugs, or anti-interleukin-6 drugs were applied in patients after lung transplantation. Additional analysis of laboratory test results for systemic inflammation and imaging studies was also carried out. The study was limited to a dedicated COVID-19 center, commonly known as a temporary hospital, and included patients infected with COVID-19 in the late post-lung transplant period (home-related infection). RESULTS: Fifteen post-lung transplantation patients with current COVID-19 infection were treated with antibodies such as tocilizumab, casirivimab, imdevimab, and regdanvimab. Of these patients, 1 was given tocilizumab (7%), 8 casirivimab and imdevimab (53%), and 2 regdanvimab (13%). Of the 15 lung transplant recipients studied, 8 presented COVID-19-associated lung changes in computed tomography scans (53%). Common clinical manifestations included dyspnea, fever, and fatigue. Antiviral agents, like remdesivir, were employed in the remaining 4 cases (27%), and adjunctive therapies, such as corticosteroids and anticoagulants, were used selectively. All treated patients survived the infection without complications; the treatment proved effective and safe.

Anti-HLA Immunization in Patients After Lung Transplantation: A Comparative Study Before and During the Pandemic.

Anti-human leukocyte antigen (anti-HLA) sensitization in lung transplant recipients (LTRs) can significantly impact graft survival and patient outcomes. The global pandemic, induced by the SARS-CoV-2 virus, brought about numerous challenges in the medical sphere, including potential alterations in HLA immunization patterns among LTRs. A retrospective analysis of LTRs group transplanted from July 2018 to 1 March 2020 (pre-pandemic) was compared with patients transplanted from 1 March 2020 to December 2022 (during the pandemic). Totally 92 patients were controlled. Patients were also divided into 2 groups: vaccinated and non-vaccinated. The results of cytotoxic crossmatch, results of anti-HLA antibody testing, presence of DSA before and after transplantation, and early and late graft function were compared between groups. In the pandemic and vaccinated groups, an increase was observed in the number of positive crossmatch tests performed with a pool of B lymphocytes. However, the presence of dithiothreitol abolished the positive reaction in 90% of cases. We also observed an increased percentage of patients immunized based on the results of solid phase tests both in the pandemic group and in the group of patients who received vaccination against the SARS-CoV-2 virus. It might be that the pandemic/vaccination has influenced the prevalence of anti-HLA immunization in LTRs. Further studies are essential to establish causative factors and develop targeted interventions for this population of patients.

Antibody-mediated rejection with detection of de novo donor-specific anti-human leukocyte antigen Class II antibodies after lung transplantation: Problems in diagnosis, treatment and monitoring on a case report basis.

Lung transplantation, like other transplants, carries a risk of graft rejection due to genetic differences between the donor and the recipient. In this paper, we focus on antibody-mediated rejection, which can cause acute and more importantly chronic graft dysfunction and subsequently shortened allograft survival. We present the case of a 46-year-old patient who, two months after lung transplantation (LTx), developed AMR manifested by the deterioration of graft function and de novo production of donor-specific antibodies (DSA): DQ3 (DQ7, DQ8, DQ9). As the patient was after left single LTx and heavily oxygen dependent a transbronchial biopsy was deemed to be high risk and it was decided to determine the clinical significance of the detected antibodies by their ability to bind complement. The test confirmed that the detected DSAs have the ability cause cytotoxicity of the transplanted organ. After treatment with methotrexate, intravenous immunoglobulin G (IVIg) and alemtuzumab, the patient's condition improved and a complete decrease in DSA was obtained. However, after a year, the production of antibodies increased sharply. Treatment with IVIg, cyclophosphamide and plasmapheresis slightly improved the patient's condition, reducing the MFI DSA values by half, but leaving them at high levels. Based on this clinical case, we discuss problems with making a diagnosis, choosing the right AMR treatment and monitoring the patient's condition during treatment. We also indicate a poor prognosis in the case of the production of DSA antibodies at the DQ locus.

Local T cell infiltrates are predominantly associated with corneal allograft rejection.

BACKGROUND: Corneal transplantations (CTXs) are a vision-saving procedure. Routinely, while CTXs' survival rates remain high, the risk of graft failure increases significantly for repeated CTXs. The reason is an alloimmunization following previous CTXs and development of memory T (Tm) and B (Bm) cells. METHODS: We characterized populations of cells present in explanted human corneas from patients receiving the first CTX and marked as a primary CTX (PCTX) or the second or more CTXs and marked as a repeated CTX (RCTX). Cells extracted from resected corneas and from peripheral blood mononuclear cells (PBMCs) were analyzed by the flow cytometry method using multiple surface and intracellular markers. RESULTS: Overall, the number of cells was similar in PCTX and RCTX patients. Extracted infiltrates from PCTXs and RCTXs contained similar numbers of T cell subsets, namely CD4+, CD8+, CD4+ Tm, CD8+ Tm, CD4+Foxp3+ T regulatory (Tregs), CD8+ Treg cells, while very few B cells (all p = NS). However, when compared to peripheral blood, PCTX and RCTX corneas contained significantly higher percentages of effector memory CD4+ and CD8+ T cells (both p < 0,05). In comparison to PCTX, RCTX group had the highest levels of Foxp3 in T CD4+ Tregs (p = 0,04) but decreased percentage of Helios-positive CD4+ Tregs. CONCLUSION: PCTXs and especially RCTXs are rejected mainly by local T cells. The accumulation of effector CD4+ and CD8+ T cells, as well as CD4+ and CD8+ Tm cells is associated with the final rejection. Furthermore, local CD4+ and CD8+ Tregs expressing Foxp3 and Helios are probably insufficient to impose the acceptance of CTX.

In-Depth Analysis of Anti-HLA Antibodies Using C1q Assay.

Alloantibodies are significant biomarkers of posttransplant kidney rejection. With solid-phase assays, the presence of alloantibodies and complement-binding capacities can be tested. It has been noted that complement-binding anti-HLA (C1q) correlates well with high titers of anti-HLA antibodies (single antigen bead, SAB), but we have recently shown that lower SAB titers may be associated with complement in the complement cytotoxicity test. If so, low titers of donor-specific antibodies could be stratified for complement binding and used for better donor-recipient matching. To study this, we tested 268 patients awaiting kidney transplantation for SAB and C1q and stratified them into positive (Allo+) and negative (Allo-) cohorts. Next, all assayed specificities of SAB were matched with the corresponding C1q in order to correlate mean fluorescence intensity levels. We found a strong correlation between SAB and C1q for all HLA loci apart from HLA-DP. Moreover, there was no strict cutoff for C1q prediction on SAB mean fluorescence intensity level. In addition, an unusual laboratory phenotype was found; that is, a positive C1q result without corresponding SAB specificity. We tested this phenomenon and found positive IgM SAB. CONCLUSIONS: Simultaneous C1q and SAB testing may serve as a tool for in-depth analysis of alloantibodies before transplantation.

Anti-HLA immunization of patients qualified for lung transplantation - Single center study.

For lung transplantation, the presence of donor-specific anti-HLA antibodies (DSA) is an important factor of antibody-mediated rejection (AMR) in its hyperacute, acute or chronic form during long-term follow up. The aim of the study was to assess the allosensitization of Polish patients qualified for a lung transplantation in our center. A retrospective study of 161 potential lung allograft recipients, also of 31 patients transplanted in the University Hospital of Gdansk, between June 2018 and December 2020 were performed. 121 potential recipients were thoroughly tested for immunization status before eventual lung transplantation. SAB-testing, PRA-CDC and vPRA assessment, and HLA typing were performed to guide donor-recipient matching and risk stratification. Then 73 patients were separated and qualified for the list of patients awaiting lung transplantation. Then 31 patients were transplanted based on a negative biological crossmatch result. The patients were generally not sensitized, as the median PRA-CDC was 0% (min 0; max 53), and the vPRA, calculated according to HLA ABDR (>2000 cut-off MFI), was 8% (min 0; max 99). If the cut-off was split into 2000 MFI for HLA ABDR, 10,000 MFI for HLAC, and 7000 MFI for HLA-DQ, the vPRA increased to 20% (min 0; max 99). The immunization status was assessed with single antigen-SAB assays. For class I, the number of any detectable alloantibodies was 14 (11.6%) 21 (17.35%) 16 (13.22%) for locus HLA-A/B/C, and 28 (23.14%) 30 (24.8%) 24 (19.8%) for locus HLA-DR/DQ/DP, respectively. The immunization of the transplanted patients was then analyzed in detail. Summarizing, the study is an analysis of the degree of anti-HLA immunization in the population of patients eligible for lung transplantation, which showed that this degree is of low intensity and can be effectively and safely and very precisely diagnosed before transplantation.

CD5-Positive B Lymphocytes after Kidney Transplantation.

Kidney transplantation is the treatment of choice for end-stage kidney diseases. Unfortunately, kidney allograft recipients rarely develop tolerance or accommodation and require life-long immunosuppression. Among many other regulatory mechanisms, CD5+ B lymphocytes (mainly B-1a) seem to be involved in the process of allograft acceptance. These cells are the major source of natural, low-affinity antibodies, which are polyreactive. Thus, we hypothesized that CD5+ B cells could be referred to as a biomarker in those patients who developed accommodation towards kidney allotransplant. In this study, 52 low-immunized kidney transplant recipients were evaluated for transplant outcome up to 8 y post-transplant. The follow up included anti-HLA antibodies, B cells phenotype and cytokines. We have identified a cohort of recipients who produced alloantibodies (Abs+), which was associated with increased levels of CD5+ B cells, mainly during the first year after transplantation but also later on. Importantly, creatinine levels were comparable between Abs+ and Abs- allorecipients at 2 years after the transplantation and graft survival rate was comparable between these groups even eight years post-transplant. So, it seems that despite the presence of alloantibodies the graft function was sustained when the level of CD5+ B cells was increased. Targeting CD5+ B cells may be a valuable therapeutic option to increase transplant success. The phenotype can be also tried as a biomarker to increase the effectiveness of individualized post-transplant treatments.

KIR Receptors as Key Regulators of NK Cells Activity in Health and Disease.

Natural killer (NK) cells are part of the cellular immune response. They target mainly cancer and virally infected cells. To a high extent cytotoxic activity of NK cells is regulated inter alia by signals from killer immunoglobulin-like receptors (KIR). The major histocompatibility complex (MHC) class I molecules are important ligands for KIR receptors. Binding of ligands (such as MHC I) to the KIR receptors has the important role in solid organ or hematopoietic cell transplantation. Of note, the understanding of the relationship between KIR and MHC receptors may contribute to the improvement of transplant results. Donor-recipient matching, which also includes the KIR typing, may improve monitoring, individualize the treatment and allow for predicting possible effects after transplantation, such as the graft-versus-leukemia effect (GvL) or viral re-infection. There are also less evident implications of KIR/MHC matching, such as with pregnancy and cancer. In this review, we present the most relevant literature reports on the importance of the KIR/MHC relationship on NK cell activity and hematopoietic stem cell transplantation (HSCT)/solid organ transplantation (SOT) effects, the risk of allograft rejection, protection against post-transplant cytomegalovirus (CMV) infection, pregnancy complications, cancer and adoptive therapy with NK cells.

Immune Algorithm Optimization for Organ Transplantation in Poland.

The lack of a uniform method for determining unacceptable HLA mismatches (UAMs) for organ transplantation worldwide has resulted in many different algorithms for donor-recipient matching. Here we present our proposal for changes to the current algorithm for immune evaluation of potential kidney recipients in Poland based on the experience of various transplantation centers. The most important finding of this article is an algorithm that stratifies the pretransplant immunologic risk based on strict laboratory criteria, enabling harmonization between transplant centers in Poland. This is because of a step-by-step algorithm for alloantibody assessment using solid-phase assays (SPA) and clearly defined technical issues, as well as cutoffs for reporting UAMs. Our novel approach focuses on a laboratory testing extension in the scope of HLA typing; detection and characterization of alloantibodies before transplantation; desensitization; and post-transplant monitoring. The proposed changes will allow for the assessment of clinically relevant anti-HLA antibodies with complement binding properties; the determination of UAMs in the potential donor; the calculation of virtual panel reactive antibodies (vPRA); the calculation of the recipient's immunologic rejection risk stratification; the assessment of the donor-recipient virtual cross-match (vXM); and the determination of the final recipient's selection for the biological cross-match testing. Collectively, the optimized algorithm permit for UAM verification is based on laboratory proofed data and will firmly improve organ allocation and transplant outcomes in Poland. We hope that this novel approach also improves the individual patient's risk stratification and future personalized treatment.

Proinsulin-specific T regulatory cells may control immune responses in type 1 diabetes: implications for adoptive therapy.

OBJECTIVE: Here we looked for possible mechanisms regulating the progression of type 1 diabetes mellitus (T1DM). In this disease, autoaggressive T cells (T conventional cells, Tconvs) not properly controlled by T regulatory cells (Tregs) destroy pancreatic islets. RESEARCH DESIGN AND METHODS: We compared the T-cell compartment of patients with newly diagnosed T1DM (NDT1DM) with long-duration T1DM (LDT1DM) ones. The third group consisted of patients with LDT1DM treated previously with polyclonal Tregs (LDT1DM with Tregs). We have also looked if the differences might be dependent on the antigen specificity of Tregs expanded for clinical use and autologous sentinel Tconvs. RESULTS: Patients with LDT1DM were characterized by T-cell immunosenescence-like changes and expansion of similar vß/T-cell receptor (TCR) clones in Tconvs and Tregs. The treatment with Tregs was associated with some inhibition of these effects. Patients with LDT1DM possessed an increased percentage of various proinsulin-specific T cells but not GAD65-specific ones. The percentages of all antigen-specific subsets were higher in the expansion cultures than in the peripheral blood. The proliferation was more intense in proinsulin-specific Tconvs than in specific Tregs but the levels of some proinsulin-specific Tregs were exceptionally high at baseline and remained higher in the expanded clinical product than the levels of respective Tconvs in sentinel cultures. CONCLUSIONS: T1DM is associated with immunosenescence-like changes and reduced diversity of T-cell clones. Preferential expansion of the same TCR families in both Tconvs and Tregs suggests a common trigger/autoantigen responsible. Interestingly, the therapy with polyclonal Tregs was associated with some inhibition of these effects. Proinsulin-specific Tregs appeared to be dominant in the immune responses in patients with T1DM and probably associated with better control over respective autoimmune Tconvs. TRIAL REGISTRATION NUMBER: EudraCT 2014-004319-35.

Association of cytokine gene polymorphisms with the complications of allogeneic haematopoietic stem cell transplantation.

The purpose of our study was to confirm the prevalence of the association between single nucleotide polymorphisms present in genes encoding cytokines and the complications occurring after haematopoietic stem cell transplantation (HSCT). 108 recipients and 81 donors were typed for TNF-α (-308), TGF-ß1 (codon 10, 25), IL-10 (-1082, -819, -592), IL-6 (-174) and INF-γ (+874). Our studies have shown a tendency toward association between the occurrence of acute form of graft versus host disease (aGVHD) and IL-6 genotype. Homozygote C/C was less likely to develop aGVHD (p=0,09). Genotype GCC/ATA in IL-10 recipient gene alone had protective effect against the occurrence of aGVHD (p=0,01). Furthermore, GCC/ATA protected the host against developing the disease in the clinically relevant grades (II-IV) (p=0,03). In addition, the recipient's T/T G/G genotype (TGF-ß1) predisposed to the development of both acute (p=0,06 - trend) and chronic (p=0,04) GVHD and also severe aGVHD (p=0,004). We also observed a statistically significant association between the genotype of recipient and the risk of infection - the protective function of the G/C IL-6 in the bloodstream infections (p=0,001). Our results suggest that IL-6, IL-10 and TGF-ß1 genotypes of recipient are the most associated with the risk of complications after HSCT.

Identification of patients with increased immunological risk among potential kidney recipients in the Polish population.

Pretransplant identification of allosensitized patients is possible thanks to new technologies, which allow for accurate detection of clinically relevant alloantibodies. Implementation of these methods in the screening of patients awaiting transplantation increased their chance for successful donor-recipient matching. Here, 1460 patients reported to the Polish National Waiting List were screened with the Luminex Screen (LS) solid phase test for anti-HLA antibodies. The patients with detected anti-HLA antibodies were assayed with the Luminex Single Antigen (LSA) tests in order to establish defined antigen specificity of the alloantibodies. The results were compared with data on the immunization assessed with the routine complement-dependent-cytotoxicity panel-reactive-antibody assay (PRA CDC). The study showed significantly higher sensitivity of the LS method when compared with PRA CDC. It has been shown that LSA test is a useful technique identifying the specificities of alloantibodies. In particular, LSA allowed to assess donor specific antibodies (DSA) to previous mismatches (MM) and to determine acceptable HLA mismatches of the potential donors. The introduction of solid phase tests in routine pretransplant diagnostics allowed for faster and more accurate assessment of the immunological risk of the recipients and optimal donor-recipient matching. Hence, the presented algorithm of solid phase assays has become a new standard for the identification of allosensitized patients awaiting kidney transplantation in Poland.

Treatment of graft-versus-host disease with naturally occurring T regulatory cells.

A significant body of evidence suggests that treatment with naturally occurring CD4(+)CD25(+) T regulatory cells (Tregs) is an appropriate therapy for graft-versus-host disease (GvHD). GvHD is a major complication of bone marrow transplantation in which the transplanted immune system recognizes recipient tissues as a non-self and destroys them. In many cases, this condition significantly deteriorates the quality of life of the affected patients. It is also one of the most important causes of death after bone marrow transplantation. Tregs constitute a population responsible for dominant tolerance to self-tissues in the immune system. These cells prevent autoimmune and allergic reactions and decrease the risk of rejection of allotransplants. For these reasons, Tregs are considered as a cellular drug in GvHD. The results of the first clinical trials with these cells are already available. In this review we present important experimental facts which led to the clinical use of Tregs. We then critically evaluate specific requirements for Treg therapy in GvHD and therapies with Tregs currently under clinical investigation, including our experience and future perspectives on this kind of cellular treatment.