A Novel Heterozygous Variant in AICDA Impairs Ig Class Switching and Somatic Hypermutation in Human B Cells and is Associated with Autosomal Dominant HIGM2 Syndrome.

B cells and their secreted antibodies are fundamental for host-defense against pathogens. The generation of high-affinity class switched antibodies results from both somatic hypermutation (SHM) of the immunoglobulin (Ig) variable region genes of the B-cell receptor and class switch recombination (CSR) which alters the Ig heavy chain constant region. Both of these processes are initiated by the enzyme activation-induced cytidine deaminase (AID), encoded by AICDA. Deleterious variants in AICDA are causal of hyper-IgM syndrome type 2 (HIGM2), a B-cell intrinsic primary immunodeficiency characterised by recurrent infections and low serum IgG and IgA levels. Biallelic variants affecting exons 2, 3 or 4 of AICDA have been identified that impair both CSR and SHM in patients with autosomal recessive HIGM2. Interestingly, B cells from patients with autosomal dominant HIGM2, caused by heterozygous variants (V186X, R190X) located in AICDA exon 5 encoding the nuclear export signal (NES) domain, show abolished CSR but variable SHM. We herein report the immunological and functional phenotype of two related patients presenting with common variable immunodeficiency who were found to have a novel heterozygous variant in AICDA (L189X). This variant led to a truncated AID protein lacking the last 10 amino acids of the NES at the C-terminal domain. Interestingly, patients' B cells carrying the L189X variant exhibited not only greatly impaired CSR but also SHM in vivo, as well as CSR and production of IgG and IgA in vitro. Our findings demonstrate that the NES domain of AID can be essential for SHM, as well as for CSR, thereby refining the correlation between AICDA genotype and SHM phenotype as well as broadening our understanding of the pathophysiology of HIGM disorders.

CTLA4 protects against maladaptive cytotoxicity during the differentiation of effector and follicular CD4+ T cells.

As chronic antigenic stimulation from infection and autoimmunity is a feature of primary antibody deficiency (PAD), analysis of affected patients could yield insights into T-cell differentiation and explain how environmental exposures modify clinical phenotypes conferred by single-gene defects. CD57 marks dysfunctional T cells that have differentiated after antigenic stimulation. Indeed, while circulating CD57+ CD4+ T cells are normally rare, we found that they are increased in patients with PAD and markedly increased with CTLA4 haploinsufficiency or blockade. We performed single-cell RNA-seq analysis of matched CD57+ CD4+ T cells from blood and tonsil samples. Circulating CD57+ CD4+ T cells (CD4cyt) exhibited a cytotoxic transcriptome similar to that of CD8+ effector cells, could kill B cells, and inhibited B-cell responses. CTLA4 restrained the formation of CD4cyt. While CD57 also marked an abundant subset of follicular helper T cells, which is consistent with their antigen-driven differentiation, this subset had a pre-exhaustion transcriptomic signature marked by TCF7, TOX, and ID3 expression and constitutive expression of CTLA4 and did not become cytotoxic even after CTLA4 inhibition. Thus, CD57+ CD4+ T-cell cytotoxicity and exhaustion phenotypes are compartmentalised between blood and germinal centers. CTLA4 is a key modifier of CD4+ T-cell cytotoxicity, and the pathological CD4cyt phenotype is accentuated by infection.

C3d-binding assay for the detection of complement activating HLA antibodies: A useful tool for allocation to highly sensitised recipients in the post-CDC era?

The CDC crossmatch test is being phased out in solid organ donor allocation, and standard luminex single antigen bead assays do not differentiate complement activating function of HLA antibodies. The current study investigated the LIFECODES C3d-binding assay to determine if it could accurately predict actual T and B cell CDC results in a cohort of highly sensitised patients. Nineteen serum samples from different highly sensitised solid organ patients were crossmatched against cells from 62 unique donors, with 174 total T and B cell crossmatches performed. The sera also underwent SAB assay using OLI and LC platforms, and C3d-binding assay. Complement activating ability of each unique HLA antibody specificity detected using SAB was assigned based on the actual CDC results, which was then used to determine the accuracy of the C3d-binding assay. The C3d-binding assay was found to be highly accurate, with sensitivity of 95%, specificity 89% and negative predictive value 97% for class I DSA and the T cell CDC crossmatch results. Furthermore, we found 100% accuracy for prediction of the complement activating function of HLA-C antibodies. Negative predictive value of above 90% was also found for HLA class II DSA. C3d-binding proved more accurate than virtual crossmatch alone to predict CDC results. This study confirms that the C3d-binding assay predicts actual CDC crossmatch results accurately. In particular, the high negative predictive value of the C3d-binding assay may be extremely useful to define HLA antibodies that do not activate complement in highly sensitised recipients.

A novel allocation strategy for the difficult-to-allocate donor: audit of deceased donor kidneys utilised for preemptive recipients within Western Australia.

Kidney donor allocation can occasionally be difficult in Australia given a small population spread over vast distances. Therefore, between 2017 and 2019 our service allowed transplantation from deceased donors into local (same-state) preemptive recipients, only if no well-matched dialysis-dependent transplant waitlist recipient was available. Transplantation using this novel allocation pathway was associated with good clinical and immunological outcomes.

Protective HLA-B57: T cell and natural killer cell recognition in HIV infection.

Understanding the basis of the immune determinants controlling disease outcome is critical to provide better care to patients and could be exploited for therapeutics and vaccine design. The discovery of the human immunodeficiency virus (HIV) virus as the causing agent of acquired immunodeficiency syndrome (AIDS) decades ago, led to a tremendous amount of research. Among the findings, it was discovered that some rare HIV+ individuals, called HIV controllers (HICs), had the ability to control the virus and keep a low viral load without the need of treatment. This ability allows HICs to delay or avoid progression to AIDS. HIV control is strongly associated with the expression of human leukocyte antigen (HLA) alleles in HICs. From the HIV protective HLAs described, HLA-B57 is the most frequent in HIC patients. HLA-B57 can present a large range of highly conserved Gag-derived HIV peptides to CD8+ T cells and natural killer (NK) cells, both the focus of this review. So far there are limited differences in the immune response strength, magnitude, or receptor repertoire towards HIV epitopes that could explain viral control in HICs. Interestingly, some studies revealed that during early infection the large breadth of the immune response towards HIV mutants in HLA-B57+ HIC patients, might in turn influence the disease outcome.

A Novel Targeted Amplicon Next-Generation Sequencing Gene Panel for the Diagnosis of Common Variable Immunodeficiency Has a High Diagnostic Yield: Results from the Perth CVID Cohort Study.

With the advent of next-generation sequencing (NGS), monogenic forms of common variable immunodeficiency (CVID) have been increasingly described. Our study aimed to identify disease-causing variants in a Western Australian CVID cohort using a novel targeted NGS panel. Targeted amplicon NGS was performed on 22 unrelated subjects who met the formal European Society for Immunodeficiencies-Pan-American Group for Immunodeficiency diagnostic criteria for CVID and had at least one of the following additional criteria: disease onset at age <18 years, autoimmunity, low memory B lymphocytes, family history, and/or history of lymphoproliferation. Candidate variants were assessed by in silico predictions of deleteriousness, comparison to the literature, and classified according to the American College of Medical Genetics and Genomics-Association for Molecular Pathology criteria. All detected genetic variants were verified independently by an external laboratory, and additional functional studies were performed if required. Pathogenic or likely pathogenic variants were detected in 6 of 22 (27%) patients. Monoallelic variants of uncertain significance were also identified in a further 4 of 22 patients (18%). Pathogenic variants, likely pathogenic variants, or variants of uncertain significance were found in TNFRSF13B, TNFRSF13C, ICOS, AICDA, IL21R, NFKB2, and CD40LG, including novel variants and variants with unexpected inheritance pattern. Targeted amplicon NGS is an effective tool to identify monogenic disease-causing variants in CVID, and is comparable or superior to other NGS methods. Moreover, targeted amplicon NGS identified patients who may benefit from targeted therapeutic strategies and had important implications for family members.

Different eplet software programs give discordant and incorrect results: An analysis of HLAMatchmaker vs Fusion Matchmaker Eplet calling software.

HLA eplet matching is a novel approach to define acceptable HLA mismatches for transplant recipients. We performed an eplet analysis of three different transplant case-series to determine if the available software programs gave accurate results. Eplet analysis was performed for three different transplant case-series typed by NGS for all HLA class I and II loci. The three different HLA datasets were entered into both the HLAMatchmaker program (v2.1) and OLI Fusion MatchMaker (v4.2) software tools. Eplet results which were discordant were cross referenced against eplet registry and published HLA allele sequence data to determine the correct assignments. The comparison reveals that there was poor concordance between the two eplet programs. Analysis of the same donor/recipient pair often gave rise to different total eplet scores, incorrect eplet mismatches and antibody verification status, and both programs have eplets assigned to incorrect HLA alleles. Overall, the OLI Fusion MatchMaker eplet tool gave more accurate and useful eplet results. Eplet matching is still primarily a research tool. Before eplet matching can enter routine clinical practice further work is required to validate the accuracy of available eplet software programs. Incorrect eplet assignment could have serious adverse consequences in the clinical transplant setting.

Preferential HLA-B27 Allorecognition Displayed by Multiple Cross-Reactive Antiviral CD8+ T Cell Receptors.

T cells provide essential immunosurveillance to combat and eliminate infection from pathogens, yet these cells can also induce unwanted immune responses via T cell receptor (TCR) cross-reactivity, also known as heterologous immunity. Indeed, pathogen-induced TCR cross-reactivity has shown to be a common, robust, and functionally potent mechanism that can trigger a spectrum of human immunopathologies associated with either transplant rejection, drug allergy, and autoimmunity. Here, we report that several virus-specific CD8+ T cells directed against peptides derived from chronic viruses (EBV, CMV, and HIV-1) presented by high frequency HLA-A and -B allomorphs differentially cross-react toward HLA-B27 allotypes in a highly focused and hierarchical manner. Given the commonality of cross-reactive T cells and their potential contribution to adverse outcomes in allogeneic transplants, our study demonstrates that multiple antiviral T cells recognizing the same HLA allomorph could pose an extra layer of complexity for organ matching.

Drug-induced alloreactivity: A new paradigm for allorecognition.

Abacavir administration is associated with drug-induced hypersensitivity reactions in HIV+ individuals expressing the HLA-B*57:01 allele. However, the immunological effects of abacavir administration in an HLA-B57 mismatched transplantation setting have not been studied. We hypothesized that abacavir exposure could induce de novo HLA-B57-specific allorecognition. HIV-specific CD8 T cell clones were generated from HIV+ individuals, using single cell sorting based on HIV peptide/HLA tetramer staining. The T cell clones were assayed for alloreactivity against a panel of single HLA-expressing cell lines, in the presence or absence of abacavir. Cytokine assay, CD137 upregulation, and cytotoxicity were used as readout. Abacavir exposure can induce de novo HLA-B57 allorecognition by HIV-specific T cells. A HIV Gag RK9/HLA-A3-specific T cell did exhibit interferon-γ production, CD137 upregulation, and cytolytic effector function against allogeneic HLA-B57, but only in the presence of abacavir. Allorecognition was specific to the virus specificity, HLA restriction, and T cell receptor TRBV use of the T cell. We provide proof-of-principle evidence that administration of a drug could induce specific allorecognition of mismatched HLA molecules in the transplant setting. We suggest that HIV-seropositive recipients of an HLA-B57 mismatched graft should not receive abacavir until further studies are completed.

Stimulation of HIV-specific T cell clonotypes using allogeneic HLA.

We hypothesized that HIV-specific CD8 T cell clonotypes can be stimulated by allogeneic HLA molecules. Multiple HIV-specific CD8 T cell clones were derived from 12 individuals with chronic HIV infection, specific for 13 different HIV Gag antigens and restricted to 7 different HLA molecules. The generated T cell clones were assayed for alloreactivity against a panel of single HLA class I expressing cell lines (SALs). HIV-specific T cells recognising at least one allogeneic HLA molecule could be identified from 7 of 12 patients tested. Allorecognition was associated with IFNγ cytokine production, CD137 upregulation and cytotoxicity, suggesting high avidity allo-stimulation. Allo-HLA recognition by HIV-specific T cells was specific to the HIV target peptide/HLA restriction and TCR TRBV usage of the T cells. HIV-specific T cells do crossreact against allogeneic HLA molecules in an epitope and TRBV specific manner. Therefore allo-HLA stimulation could be exploited to induce or augment HIV-specific T cell responses.

Peri-operative third party red blood cell transfusion in renal transplantation and the risk of antibody-mediated rejection and graft loss.

Historic red blood cell transfusion (RBCT) may induce anti-HLA antibody which, if donor specific (DSA), is associated with increased antibody-mediated rejection (AMR). Whether post-operative RBCT influences this risk is unknown. We examined the RBCT history in 258 renal transplant recipients stratified according to prevalent recipient HLA antibody (DSA, Non-DSA or No Antibody). AMR occurred more frequently in patients who received RBCT both pre and post transplant compared with all other groups (Pre+Post-RBCT 21%, Pre-RBCT 4%, Post-RBCT 6%, No-RBCT 6%, HR 4.1 p=0.004). In the 63 patients who received Pre+Post-RBCT, 65% (13/20) with DSA developed AMR compared with 0/6 in the Non-DSA group and 2/37 (5%) in the No-Antibody group (HR 13.9 p<0.001). In patients who received No-RBCT, Pre-RBCT or Post-RBCT there was no difference in AMR between patients with DSA, Non-DSA or No-Antibody. Graft loss was independently associated with Pre+Post-RBCT (HR 6.5, p=0.001) AMR (HR 23.9 p<0.001) and Non-AMR (6.0 p=0.003) after adjusting for DSA and delayed graft function. Re-exposure to RBCT at the time of transplant is associated with increased AMR only in patients with preformed DSA, suggesting that RBCT provides additional allostimulation. Patients receiving Pre+Post-RBCT also had an increased risk of graft loss independently of AMR or DSA. Both pre and post procedural RBCT in renal transplantation is associated with modification of immunological risk and warrants additional study.

Stimulation of human EBV- and CMV-specific cytolytic effector function using allogeneic HLA molecules.

Viral infection is a major cause of morbidity and mortality, and there are few therapeutic options available to augment a virus-specific T cell response. Although allo-HLA cross-reactivity from virus-specific memory T cells is common, it is unclear whether priming with specific allogeneic cells could conversely elicit a viral peptide/self-HLA restricted cytotoxic T cell response in humans. First, we used the previously described allo-HLA-B*44:02 cross-reactivity of EBV peptide/HLA-B8 restricted T cells, to determine whether allogeneic HLA stimulation can elicit a cytolytic immune response against EBV. HLA-B8(+) HLA-B44(-) EBV-seropositive PBMCs were stimulated with either HLA-B*44:02(+) or HLA-B*44:03(+) mismatched irradiated PBMCs in a 7-10 d MLR. The allo-HLA stimulated responder cells were then evaluated for cytotoxicity using EBV peptide loaded autologous target cells and unloaded HLA-B8(+) EBV LCL target cells. PBMCs from EBV-seropositive donors gained EBV-specific cytolytic effector function following specific allo-HLA stimulation. Finally, we also elicited cytolytic CMV-specific responses using specific allogeneic cell stimulation, to confirm that this technique can be used to elicit viral peptide/self-HLA restricted responses even from nonpublic TCR responses. Allogeneic cell stimulation used as a cell therapy may be a potential tool to augment an antiviral T cell response in patients with EBV or CMV infection.

Detection of allo-HLA cross-reactivity by virus-specific memory T-cell clones using single HLA-transfected K562 cells.

The ability to directly measure virus-specific lymphocytes using fluorochrome-labeled tetrameric complexes has proven a great advancement for the transplantation field. Viral peptide/HLA tetrameric complexes allow the rapid generation of virus-specific clones using single cell sorting apparatus, permitting the determination of alloreactivity from a single TCR with known specificity. When combined with new target "detector" cells called single HLA antigen-transfected K562 cells (SALs), the human alloresponse can for the first time be examined specifically and reliably. Here we describe a method for detection of "heterologous immunity" from virus-specific memory T-cells using single HLA expressing cell lines as allogeneic targets.

Antibody and B-cell responses may control circulating lipopolysaccharide in patients with HIV infection.

OBJECTIVES: To examine the relationship between plasma markers of microbial translocation and antibodies to lipopolysaccharide (LPS) and circulating memory B cells in patients with HIV infection. DESIGN: Cross-sectional study in antiretroviral therapy (ART)-naive (n = 23) and ART-treated (n = 27) HIV patients. METHODS: Antibodies to LPS and immunoglobulins, assayed in stored serum, and matched memory B-cell counts were correlated with levels of LPS and bacterial 16S ribosome DNA (16S rDNA), assayed in stored plasma. RESULTS: In ART-naive patients, plasma LPS levels correlated inversely with serum levels of IgG and IgA antibodies to LPS (P = 0.03 and 0.006, respectively), serum levels of IgA anti-LPS correlated with total IgA (P < 0.0001) and levels of IgG anti-LPS correlated with IgM(+) memory B-cell counts (P = 0.025). In ART-treated patients, plasma LPS levels were not related to levels of LPS antibodies, but were related to CD4(+) T-cell and switched memory B-cell counts. There were no correlations with plasma levels of 16S rDNA. CONCLUSION: Plasma LPS levels were associated with antibody and possibly B-cell responses to LPS in ART-naive HIV patients, whereas they were associated with the degree of immune reconstitution in ART-treated patients.

Audit of emergency department assessment and management of patients presenting with community-acquired needle stick injuries.

OBJECTIVES: To describe characteristics and management of people with community acquired needle stick injuries (CANSI) attending urban emergency departments; and suggest a guideline to improve assessment, management, and documentation. METHODS: A retrospective analysis of cases with CANSI attending emergency departments in two tertiary hospitals between 2001 and 2005 using medical record review with follow up phone and written survey. RESULTS: Thirty-nine cases met the criteria for CANSI. Persons younger than 30 years sustained 48.72% of all injuries. Source serology was available for only five cases (12.82%). Thirty-one of thirty-nine patients (79.49%) were classed as not immune to hepatitis B but only four of these (12.90%) received both hepatitis B vaccination and hepatitis B immunoglobulin. Six patients (15.38%) received HIV prophylaxis; of which two (33.33%) did not receive baseline HIV testing. Of ten patients referred to immunology clinic for follow up only two (20.00%) attended at 6 months. CONCLUSION: We have identified groups that are at high risk of CANSI, including young males, security workers and cleaners. In the majority of cases protection against hepatitis B was inadequately provided, and a substantial proportion had inadequate baseline assessment and documentation. A guideline is suggested that may be used to improve these deficits.

Vaccine-induced allo-HLA-reactive memory T cells in a kidney transplantation candidate.

BACKGROUND: Allo-human leukocyte antigen (HLA) reactivity by naturally acquired viral-specific memory T cells is common. However, the effect of successful vaccination on the alloreactive memory T-cell repertoire is unclear. We hypothesized that vaccination could specifically induce allo-HLA-reactive memory T cells. METHODS: A varicella-zoster virus (VZV) immediate early 62 (IE62)-specific CD8 memory T-cell clone was single cell sorted from a VZV seronegative renal transplant candidate after response to live attenuated varicella vaccination. To analyze the allo-HLA reactivity, the VZV IE62-specific T-cell clone was tested against HLA-typed target cells and target cells transfected with HLA molecules, in both cytokine production and cytotoxicity assays. RESULTS: The varicella vaccine-induced VZV IE62-specific T-cell clone specifically produced interferon-γ when stimulated with HLA-B*55:01-expressing Epstein-Barr virus-transformed B cells and HLA-B*55:01-transfected K562 cells (single HLA antigen expressing cell line [SALs]) only. The clone also demonstrated specific cytolytic effector function against HLA-B*55:01 SALs and phytohemagglutinin blasts. Cytotoxicity assays using proximal tubular epithelial cell and human umbilical vein endothelial cell targets confirmed the kidney tissue specificity of the allo-HLA-B*55:01 reactivity, and the relevance of the cross-reactivity to clinical kidney transplantation. The results also suggest that molecular mimicry, and not bystander proliferation, is the mechanism underlying vaccine-induced alloreactivity. CONCLUSIONS: Varicella vaccination generated a de novo alloreactive kidney cell-specific cytolytic effector memory T cell in a patient awaiting renal transplantation. Vaccination-induced alloreactivity may have important clinical implications, especially for vaccine timing and recipient monitoring.

Tissue specificity of cross-reactive allogeneic responses by EBV EBNA3A-specific memory T cells.

BACKGROUND: The crossreactivity of Epstein-Barr virus (EBV Epstein-Barr virus nuclear antigen 3A [EBNA3A])-specific CD8 T cells against allogeneic human leukocyte antigen (HLA)-B*44:02 has been shown to be dependent on presentation of self-peptide EEYLQAFTY by the target antigen. In this study, we report that allogeneic HLA-B*44:02 proximal tubular epithelial cells (PTECs) and human umbilical vein endothelial cells (HUVECs) are poor targets for EBV EBNA3A-specific T cells. METHODS: The EEY peptide was exogenously loaded onto HLA-B*44:02 and HLA-B*44:03-expressing PTECs and HUVECs. EEY-peptide-loaded, and unloaded, PTECs and HUVECs were then incubated with serial dilutions of our EBNA3A T-cell clone, in a cytotoxicity assay. RESULTS: Although HLA-B*44:02-expressing PTECs were specifically lysed in proportion to the effector/target ratio by the EBNA3A T-cell clone, without peptide loading, lysis was greatly increased by exogenous EEY peptide loading (15% vs. 75%; P<0.0001). HLA-B*44:02-expressing HUVECs were only lysed when loaded with exogenous EEY peptide (0% vs. 64%; P<0.0001). Lack of HLA expression and lack of ABCD3 gene expression were excluded as a cause for these results. PTECs and HUVECs were specifically targeted by another alloreactive T-cell clone without exogenous peptide loading, suggesting that the lack of recognition of HLA-B*44:02 epithelial and endothelial cells by the EBV EBNA3A T-cell clone was due to lack of EEYLQAFTY peptide presentation. CONCLUSIONS: Tissue-specific (peptide dependent) alloreactivity may have important implications for transplantation monitoring and rejection.