Inverted direct allorecognition triggers early donor-specific antibody responses after transplantation.

The generation of antibodies against donor-specific major histocompatibility complex (MHC) antigens, a type of donor-specific antibodies (DSAs), after transplantation requires that recipient's allospecific B cells receive help from T cells. The current dogma holds that this help is exclusively provided by the recipient's CD4+ T cells that recognize complexes of recipient's MHC II molecules and peptides derived from donor-specific MHC alloantigens, a process called indirect allorecognition. Here, we demonstrated that, after allogeneic heart transplantation, CD3ε knockout recipient mice lacking T cells generate a rapid, transient wave of switched alloantibodies, predominantly directed against MHC I molecules. This is due to the presence of donor CD4+ T cells within the graft that recognize intact recipient's MHC II molecules expressed by B cell receptor-activated allospecific B cells. Indirect evidence suggests that this inverted direct pathway is also operant in patients after transplantation. Resident memory donor CD4+ T cells were observed in perfusion liquids of human renal and lung grafts and acquired B cell helper functions upon in vitro stimulation. Furthermore, T follicular helper cells, specialized in helping B cells, were abundant in mucosa-associated lymphoid tissue of lung and intestinal grafts. In the latter, more graft-derived passenger T cells correlated with the detection of donor T cells in recipient's circulation; this, in turn, was associated with an early transient anti-MHC I DSA response and worse transplantation outcomes. We conclude that this inverted direct allorecognition is a possible explanation for the early transient anti-MHC DSA responses frequently observed after lung or intestinal transplantations.

Natural killer cell infiltration is discriminative for antibody-mediated rejection and predicts outcome after kidney transplantation.

Despite partial elucidation of the pathophysiology of antibody-mediated rejection (ABMR) after kidney transplantation, it remains largely unclear which of the involved immune cell types determine disease activity and outcome. We used microarray transcriptomic data from a case-control study (n=95) to identify genes that are differentially expressed in ABMR. Given the co-occurrence of ABMR and T-cell-mediated rejection (TCMR), we built a bioinformatics pipeline to distinguish ABMR-specific mRNA markers. Differential expression of 503 unique genes was identified in ABMR, with significant enrichment of natural killer (NK) cell pathways. CIBERSORT (Cell type Identification By Estimating Relative Subsets Of known RNA Transcripts) deconvolution analysis was performed to elucidate the corresponding cell subtypes and showed increased NK cell infiltration in ABMR in comparison to TCMR and normal biopsies. Other leukocyte types (including monocytes/macrophages, CD4 and CD8 T cells, and dendritic cells) were increased in rejection, but could not discriminate ABMR from TCMR. Deconvolution-based estimation of NK cell infiltration was validated using computerized morphometry, and specifically associated with glomerulitis and peritubular capillaritis. In an external data set of kidney transplant biopsies, activated NK cell infiltration best predicted graft failure amongst all immune cell subtypes and even outperformed a histologic diagnosis of acute rejection. These data suggest that NK cells play a central role in the pathophysiology of ABMR and graft failure after kidney transplantation.

CD4+ T Cell Help Is Mandatory for Naive and Memory Donor-Specific Antibody Responses: Impact of Therapeutic Immunosuppression.

Antibody-mediated rejection is currently the leading cause of transplant failure. Prevailing dogma predicts that B cells differentiate into anti-donor-specific antibody (DSA)-producing plasma cells only with the help of CD4+ T cells. Yet, previous studies have shown that dependence on helper T cells decreases when high amounts of protein antigen are recruited to the spleen, two conditions potentially met by organ transplantation. This could explain why a significant proportion of transplant recipients develop DSA despite therapeutic immunosuppression. Using murine models, we confirmed that heart transplantation, but not skin grafting, is associated with accumulation of a high quantity of alloantigens in recipients' spleen. Nevertheless, neither naive nor memory DSA responses could be observed after transplantation of an allogeneic heart into recipients genetically deficient for CD4+ T cells. These findings suggest that DSA generation rather result from insufficient blockade of the helper function of CD4+ T cells by therapeutic immunosuppression. To test this second theory, different subsets of circulating T cells: CD8+, CD4+, and T follicular helper [CD4+CXCDR5+, T follicular helper cells (Tfh)], were analyzed in 9 healthy controls and 22 renal recipients. In line with our hypothesis, we observed that triple maintenance immunosuppression (CNI + MMF + steroids) efficiently blocked activation-induced upregulation of CD25 on CD8+, but not on CD4+ T cells. Although the level of expression of CD40L and ICOS was lower on activated Tfh of immunosuppressed patients, the percentage of CD40L-expressing Tfh was the same than control patients, as was Tfh production of IL21. Induction therapy with antithymocyte globulin (ATG) resulted in prolonged depletion of Tfh and reduction of CD4+ T cells number with depleting monoclonal antibody in murine model resulted in exponential decrease in DSA titers. Furthermore, induction with ATG also had long-term beneficial influence on Tfh function after immune reconstitution. We conclude that CD4+ T cell help is mandatory for naive and memory DSA responses, making Tfh cells attractive targets for improving the prevention of DSA generation and to prolong allograft survival. Waiting for innovative treatments to be translated into the clinical field ATG induction seems to currently offer the best clinical prospect to achieve this goal.

Residual Activatability of Circulating Tfh17 Predicts Humoral Response to Thymodependent Antigens in Patients on Therapeutic Immunosuppression.

The generation of antibodies against protein antigens (such as donor-specific HLA molecules) requires that T follicular helper cells (Tfh) provide help to B cells. Immunosuppressive (IS) armamentarium prevents T cell activation, yet a significant proportion of renal transplant patients develop donor-specific antibodies (DSA), which suggests that IS drugs do not efficiently block T follicular helper cells. To test this hypothesis, the number of circulating Tfh, their polarization profile, and ability to up-regulate (i) the co-stimulatory molecules CD40L and ICOS, and (ii) the activation marker CD25, following in vitro stimulation in presence of IS drugs, were compared between 36 renal transplant patients (6-72 months post transplantation) and nine healthy controls. IS drugs reduced the number of Tfh1 and 2 but had little impact on Tfh17, which was the dominant subset in transplant patients. Although, IS drugs decreased activation-induced expression of co-stimulatory molecules by Tfh, the impact was highly variable between individuals. Furthermore, 20% of transplant patients displayed normal expression of CD25 on Tfh following in vitro stimulation (i.e., "residual activatability"). To test whether residual activatability of Tfh correlates with antibody response against thymo-dependent antigens we took advantage of the 2015 influenza vaccination campaign, which provided a normalized setting for antigenic stimulation. In line with our hypothesis, responders to influenza vaccine exhibited significantly higher percentage of CD25-expressing Tfh17 after in vitro stimulation. A results that was confirmed retrospectively in nine transplanted patients at the time of first DSA detection. We concluded that "residual activatability" of Tfh17 might be used as a non-invasive biomarker to identify transplant patients at higher risk to develop DSA under immunosuppression. If validated in larger studies, this assay might help optimizing the prevention of DSA through personalized adaptation of immunosuppressive regimen.

Lack of the nucleoside transporter ENT1 results in the Augustine-null blood type and ectopic mineralization.

The Augustine-negative alias At(a-) blood type, which seems to be restricted to people of African ancestry, was identified half a century ago but remains one of the last blood types with no known genetic basis. Here we report that a nonsynonymous single nucleotide polymorphism in SLC29A1 (rs45458701) is responsible for the At(a-) blood type. The resulting p.Glu391Lys variation in the last extracellular loop of the equilibrative nucleoside transporter 1 (ENT1; also called SLC29a1) is known not to alter its ability to transport nucleosides and nucleoside analog drugs. Furthermore, we identified 3 individuals of European ancestry who are homozygous for a null mutation in SLC29A1 (c.589+1G>C) and thus have the Augustine-null blood type. These individuals lacking ENT1 exhibit periarticular and ectopic mineralization, which confirms an important role for ENT1/SLC29A1 in human bone homeostasis as recently suggested by the skeletal phenotype of aging Slc29a1(-/-) mice. Our results establish Augustine as a new blood group system and place SLC29A1 as a new candidate gene for idiopathic disorders characterized with ectopic calcification/mineralization.

Family study of a Swiss patient uncovered a novel genetic basis for the S-s-U+(var) phenotype.

BACKGROUND: The rare S-s- phenotype is typically found in persons of African origin. Three genetic bases underlying this phenotype have been identified so far: a large deletion including the GYPB gene, which encodes the S and s antigens, and two mutations affecting GYPB splicing (commonly called "P2" and "NY"). The discovery of the S-s- phenotype in a Swiss patient prompted this study. STUDY DESIGN AND METHODS: The GYPB genotype of the patient was analyzed with Beadchip technology and Sanger sequencing. GYPB haplotype analysis was also carried out in the patient's family. A functional splicing assay was developed to determine the impact of the identified mutation on GYPB splicing. RESULTS: Sanger sequencing of GYPB in the patient indicated that she was homozygous for a GYPB*s allele carrying a novel mutation in the splice donor site of Intron 5 (c.270+5G>A). Analysis of GYPB haplotypes in the patient's family revealed that she actually inherited this mutated GYPB*s allele from her mother of Swiss ancestry and a deleted GYPB allele from her father of Egyptian ancestry. Using a minigene-based splicing assay, we showed that GYPB mutation c.270+5G>A causes the skipping of Exon B5, as previously reported for the P2 mutation (c.270+5G>T). Consistently, the patient's red blood cells were found to be S-s-U+(var) . CONCLUSION: A novel GYPB mutation (c.270+5G>A) accounting for the S-s-U+(var) phenotype was identified. In contrast with P2 and NY mutations, which also drive this rare phenotype, this novel GYPB mutation inactivates a GYPB*s allele and does not appear to be of African origin.

Lethal autoimmune hemagglutination due to an immunoglobulin A autoagglutinin with Band 3 specificity.

BACKGROUND: We describe a patient with a high-titer warm immunoglobulin (Ig)A autoantibody resulting in death due to hemagglutination rather than to hemolysis. CASE REPORT: A 47-year-old male patient presented with an intriguing pronounced vascular erythema of the skin. A livedo reticularis associated with cold agglutinin of high thermal amplitude was suspected. The patient's condition unexpectedly and abruptly deteriorated resulting in death 3 days after admission. STUDY DESIGN AND METHODS: Conventional serologic procedures and immunochemical methods were used. RESULTS: Serologic and immunochemical examinations revealed a warm IgA autoantibody of high titer with anti-Band 3 specificity. Although the patient presented with severe anemia, only mild signs of hemolysis were observed, with no evidence of complement activation. The autopsy revealed an enormous accumulation of agglutinated red blood cells in liver and spleen and a B-cell lymphoma and cerebral edema. Thus, the patient's death was largely caused by hypoxia related to hemagglutination rather than to hemolysis and/or anemia per se. CONCLUSION: Strongly hemagglutinating antibodies may not only cause immune hemolysis but also hypoxia due to intravascular hemagglutination.

Disruption of SMIM1 causes the Vel- blood type.

Here, we report the biochemical and genetic basis of the Vel blood group antigen, which has been a vexing mystery for decades, especially as anti-Vel regularly causes severe haemolytic transfusion reactions. The protein carrying the Vel blood group antigen was biochemically purified from red blood cell membranes. Mass spectrometry-based de novo peptide sequencing identified this protein to be small integral membrane protein 1 (SMIM1), a previously uncharacterized single-pass membrane protein. Expression of SMIM1 cDNA in Vel- cultured cells generated anti-Vel cell surface reactivity, confirming that SMIM1 encoded the Vel blood group antigen. A cohort of 70 Vel- individuals was found to be uniformly homozygous for a 17 nucleotide deletion in the coding sequence of SMIM1. The genetic homogeneity of the Vel- blood type, likely having a common origin, facilitated the development of two highly specific DNA-based tests for rapid Vel genotyping, which can be easily integrated into blood group genotyping platforms. These results answer a 60-year-old riddle and provide tools of immediate assistance to all clinicians involved in the care of Vel- patients.

Molecular analysis of the rare in(Lu) blood type: toward decoding the phenotypic outcome of haploinsufficiency for the transcription factor KLF1.

KLF1 encodes an erythroid transcription factor, whose essential function in erythropoiesis has been demonstrated by extensive studies in mouse models. The first reported mutations in human KLF1 were found in individuals with a rare and asymptomatic blood type called In(Lu). Here, we show that KLF1 haploinsufficiency is responsible for the In(Lu) blood type, after redefining this peculiar blood type using flow cytometry to quantify the levels of BCAM and CD44 on red blood cells. We found 10 (seven novel) heterozygous KLF1 mutations responsible for the In(Lu) blood type. Although most were obligate loss-of-function mutations due to the truncation of the DNA-binding domain of KLF1, three were missense mutations that were located in its DNA-binding domain and impaired the transactivation capacity of KLF1 in vitro. We further showed that the levels of the hemoglobin variants HbF and HbA(2) were increased in the In(Lu) blood type, albeit differently. The levels of the membrane glycoproteins BCAM and CD44 were also differently reduced on In(Lu) red blood cells. This biochemical and genetic analysis of the In(Lu) blood type tackles the phenotypic outcome of haploinsufficiency for a transcription factor.

Null alleles of ABCG2 encoding the breast cancer resistance protein define the new blood group system Junior.

The breast cancer resistance protein, also known as ABCG2, is one of the most highly studied ATP-binding cassette (ABC) transporters because of its ability to confer multidrug resistance. The lack of information on the physiological role of ABCG2 in humans severely limits cancer chemotherapeutic approaches targeting this transporter. We report here that ABCG2 comprises the molecular basis of a new blood group system (Junior, Jr) and that individuals of the Jr(a-) blood type have inherited two null alleles of ABCG2. We identified five frameshift and three nonsense mutations in ABCG2. We also show that the prevalence of the Jr(a-) blood type in the Japanese and European Gypsy populations is related to the p.Gln126* and p.Arg236* protein alterations, respectively. The identification of ABCG2(-/-) (Jr(a-)) individuals who appear phenotypically normal is an essential step toward targeting ABCG2 in cancer and also in understanding the physiological and pharmacological roles of this promiscuous transporter in humans.

ABCB6 is dispensable for erythropoiesis and specifies the new blood group system Langereis.

The human ATP-binding cassette (ABC) transporter ABCB6 has been described as a mitochondrial porphyrin transporter essential for heme biosynthesis, but it is also suspected to contribute to anticancer drug resistance, as do other ABC transporters located at the plasma membrane. We identified ABCB6 as the genetic basis of the Lan blood group antigen expressed on red blood cells but also at the plasma membrane of hepatocellular carcinoma (HCC) cells, and we established that ABCB6 encodes a new blood group system (Langereis, Lan). Targeted sequencing of ABCB6 in 12 unrelated individuals of the Lan(-) blood type identified 10 different ABCB6 null mutations. This is the first report of deficient alleles of this human ABC transporter gene. Of note, Lan(-) (ABCB6(-/-)) individuals do not suffer any clinical consequences, although their deficiency in ABCB6 may place them at risk when determining drug dosage.

Anti-U-like as an alloantibody in S-s-U- and S-s-U+(var) black people.

BACKGROUND: S, s, and U antigens belong to the MNS system. They are carried by glycophorin B (GPB), encoded by GYPB. Black people with the low-prevalence S-s- phenotype, either U- or U+(var), can make a clinically significant anti-U. Anti-U-like, a cold immunoglobulin G autoantibody quite commonly observed in S-s+U+ black persons, was previously described to be nonreactive with ficin-, α-chymotrypsin-, and pronase-treated red blood cells (RBCs); nonreactive or weakly reactive with papain-treated RBCs; and reactive with trypsin-treated RBCs. Here we describe, in S-s- people from different molecular backgrounds, an alloantibody to a high-prevalence GPB antigen, which presents the same pattern of reactivity with proteases as autoanti-U-like. STUDY DESIGN AND METHODS: Four S-s- patients with an alloantibody to a high-prevalence GPB antigen were investigated by serologic and molecular methods. RESULTS: An alloantibody was observed in two S-s-U-/Del GYPB, one S-s-U+(var)/GYPB(P2), and one S-s-U+(var)/GYPB(NY) patients. As this alloantibody showed the same pattern of reactivity with proteases as autoanti-U-like, we decided to name it "anti-U-like." Anti-U-like made by the two S-s-U- patients was reactive with the S-s-U+(var) RBCs of the two other patients. CONCLUSION: S-s-U-/Del GYPB, S-s-U+(var)/GYPB(P2), and S-s-U+(var)/GYPB(NY) patients can make an alloanti-U-like. Anti-U-like made by S-s-U- people appears reactive with GYPB(P2) and GYPB(NY) RBCs, which both express a weak and partial U-like reactivity. We recommend transfusing S-s-U- RBCs in S-s-U- patients showing alloanti-U-like. Our study contributes to a better understanding of alloimmunization to GPB in black people and confirms importance of genotyping in S-s- patients, especially those with sickle cell disease to be frequently transfused.

A dominant mutation in the gene encoding the erythroid transcription factor KLF1 causes a congenital dyserythropoietic anemia.

The congenital dyserythropoietic anemias (CDAs) are inherited red blood cell disorders whose hallmarks are ineffective erythropoiesis, hemolysis, and morphological abnormalities of erythroblasts in bone marrow. We have identified a missense mutation in KLF1 of patients with a hitherto unclassified CDA. KLF1 is an erythroid transcription factor, and extensive studies in mouse models have shown that it plays a critical role in the expression of globin genes, but also in the expression of a wide spectrum of genes potentially essential for erythropoiesis. The unique features of this CDA confirm the key role of KLF1 during human erythroid differentiation. Furthermore, we show that the mutation has a dominant-negative effect on KLF1 transcriptional activity and unexpectedly abolishes the expression of the water channel AQP1 and the adhesion molecule CD44. Thus, the study of this disease-causing mutation in KLF1 provides further insights into the roles of this transcription factor during erythropoiesis in humans.