Reactivity of T cells from women with antibodies to the human platelet antigen (HPA)-1a to peptides encompassing the HPA-1 polymorphism

The human platelet antigen-1a (HPA-1a) is the most common alloantigenic target in fetal and neonatal alloimmune thrombocytopenia (NAIT). Treatment currently depends on the outcome in previous pregnancies. HPA-1 specific T cell responses were determined in 14 HPA-1a alloimmunized women during or after pregnancies affected by NAIT. Peripheral blood mononuclear cells were incubated with peptides encompassing the Leu33Pro polymorphism (residues 20-39 and 24-45 in both Leu33 (HPA-1a) and Pro33 (HPA-1b) forms) or control recall antigens in the presence of autologous sera and T cell proliferation was measured by (3)H-thymidine incorporation. Control antenatal and postpartum sera suppressed T cell proliferation and use of such sera was avoided. Most patients (86%) responded to the HPA-1a peptides with 64% also having weaker T cell proliferation to the HPA-1b peptides; 14% had no activity towards any peptide despite responding to control antigens. Administration of IVIG during pregnancy appeared to reduce T cell reactivity to HPA-1 peptides. Postnatal anti-HPA-1a T cell responses from women who had a severe history of NAIT (an intracranial haemorrhage in a previous fetus) were greater than those from women with a mild history. This assay may have the potential to predict disease severity if performed prior to or early in pregnancy.     

Clinical efficacy of a new CD28‐targeting antagonist of T cell co‐stimulation in a non‐human primate model of collagen‐induced arthritis

T cells have a central pathogenic role in the aetiopathogenesis of rheumatoid arthritis (RA), and are therefore a favoured target of immunotherapy aiming at physical or functional elimination. Here we report an efficacy test of FR104, a new co‐stimulation inhibitor directly targeting CD28 on T cells, in a translationally relevant model, the rhesus monkey model of collagen‐induced arthritis (CIA). As a relevant comparator we used abatacept [cytotoxic T lymphocyte antigen immunoglobulin (CTLA Ig)], an antagonist of CTLA‐4 binding to CD80/86 clinically approved for treatment of RA. Treatment with either compound was started at the day of CIA induction. Although FR104 previously demonstrated a higher control of T cell responses in vitro than abatacept, both compounds were equally potent in the suppression of CIA symptoms and biomarkers, such as the production of C‐reactive protein (CRP) and interleukin (IL)‐6 and anti‐collagen type II (CII) serum antibody (IgM/IgG). However, in contrast to abatacept, FR104 showed effective suppression of CII‐induced peripheral blood mononuclear cell (PBMC) proliferation. The current study demonstrates a strong potential of the new selective CD28 antagonist FR104 for treatment of RA.     

Programmed death (PD)‐1 molecule and its ligand PD‐L1 distribution among memory CD4 and CD8 T cell subsets in human immunodeficiency virus‐1‐infected individuals

Human immunodeficiency virus (HIV)‐1 causes T cell anergy and affects T cell maturation. Various mechanisms are responsible for impaired anti‐HIV‐1‐specific responses: programmed death (PD)‐1 molecule and its ligand PD‐L1 are negative regulators of T cell activity and their expression is increased during HIV‐1 infection. This study examines correlations between T cell maturation, expression of PD‐1 and PD‐L1, and the effects of their blockade. Peripheral blood mononuclear cells (PBMC) from 24 HIV‐1⁺ and 17 uninfected individuals were phenotyped for PD‐1 and PD‐L1 expression on CD4⁺ and CD8⁺ T cell subsets. The effect of PD‐1 and PD‐L1 blockade on proliferation and interferon (IFN)‐γ production was tested on eight HIV‐1⁺ patients. Naive (CCR7⁺CD45RA⁺) CD8⁺ T cells were reduced in HIV‐1 aviraemic (P = 0·0065) and viraemic patients (P = 0·0130); CD8 T effector memory subsets [CCR7^‐CD45RA^–(T_EM)] were increased in HIV‐1⁺ aviraemic (P = 0·0122) and viraemic (P = 0·0023) individuals versus controls. PD‐1 expression was increased in CD4 naive (P = 0·0496), central memory [CCR7⁺CD45RA^– (T_CM); P = 0·0116], T_EM (P = 0·0037) and CD8 naive T cells (P = 0·0133) of aviraemic HIV‐1⁺versus controls. PD‐L1 was increased in CD4 T_EMRA (CCR7^‐CD45RA⁺, P = 0·0119), CD8 T_EM (P = 0·0494) and CD8 T_EMRA (P = 0·0282) of aviraemic HIV‐1⁺versus controls. PD‐1 blockade increased HIV‐1‐specific proliferative responses in one of eight patients, whereas PD‐L1 blockade restored responses in four of eight patients, but did not increase IFN‐γ‐production. Alteration of T cell subsets, accompanied by increased PD‐1 and PD‐L1 expression in HIV‐1 infection contributes to anergy and impaired anti‐HIV‐1‐specific responses which are not rescued when PD‐1 is blocked, in contrast to when PD‐L1 is blocked, due possibly to an ability to bind to receptors other than PD‐1.     

Human leucocyte antigen‐Bw4 and Gag‐specific T cell responses are associated with slow disease progression in HIV‐1B‐infected anti‐retroviral therapy‐naive Chinese

In China, the majority of human immunodeficiency virus (HIV) infections are predominately subtype B. It is important to characterize the HIV‐1 subtype B‐specific and its T cell response within the Chinese population, with the aim of identifying protective correlates of immunity to control HIV‐1 infections. In this study, we performed a comprehensive analysis looking into the magnitude/strength of T cell responses directed at the Gag protein of the HIV‐1 subtype B, one of the most conserved HIV‐1 proteins. The study group consisted of anti‐retroviral native and chronic HIV‐1 subtype B‐infected individuals. We used enzyme‐linked immunospot (ELISPOT) assay to quantify the total T cell responses to HIV‐1 Gag at the single peptide level. Twenty‐eight (38%) peptides were recognized in 24 (82·8%) individuals. The p24 was identified as the most frequently recognized subunit protein with the greatest T cell response in the test, which correlated positively with CD4⁺ T cell count and inversely with viral load (VL). At the level of the human leucocyte antigen (HLA) supertypes, we detected the highest levels and a significant correlation with both the CD4⁺ T cell count and the VL with Gag T cell responses in Bw4/Bw4. These findings demonstrate that (i) the HIV‐1B Gag p24‐specific immune responses play an important role in controlling viral replication and slowing clinical progression; and (ii) HLA‐Bw4/Bw4 allele has stronger T cell responses, which is associated with slow clinical progression in Chinese HIV patients.     

Screening of anti‐HPA‐1a antibodies in HPA‐1a‐negative mothers

Results from a study of more than 100 000 pregnant women show that the pathophysiology of neonatal alloimmune thrombocytopenia (NAIT) and haemolytic disease of the newborn (HDN) has many equalities [ 1 ]. It has been reported that NAIT is more than three times more frequent compared with HDN. This makes it tempting to consider a design for screening for HPA 1a negativity and follow up in/after the pregnancy based on the same principles as for RhD‐negative pregnant women. Even without available vaccination, it is strongly indicated that screening and Caesarean section 2–4 weeks prior to term in mothers with the HPA‐1a‐negative platelet type, reduce neonatal morbidity and mortality due to NAIT.     

Efficient and versatile manipulation of the peripheral CD4+ T‐cell compartment by antigen targeting to DNGR‐1/CLEC9A

DC NK lectin group receptor‐1 (DNGR‐1, also known as CLEC9A) is a C‐type lectin receptor expressed by mouse CD8α⁺ DC and by their putative equivalents in human. DNGR‐1 senses necrosis and regulates CD8⁺ T‐cell cross‐priming to dead‐cell‐associated antigens. In addition, DNGR‐1 is a target for selective in vivo delivery of antigens to DC and the induction of CD8⁺ T‐cell and Ab responses. In this study, we evaluated whether DNGR‐1 targeting can be additionally used to manipulate antigen‐specific CD4⁺ T lymphocytes. Injection of small amounts of antigen‐coupled anti‐DNGR‐1 mAb into mice promoted MHC class II antigen presentation selectively by CD8α⁺ DC. In the steady state, this was sufficient to induce proliferation of antigen‐specific naïve CD4⁺ T cells and to drive their differentiation into Foxp3⁺ regulatory lymphocytes. Co‐administration of adjuvants prevented this induction of tolerance and promoted immunity. Notably, distinct adjuvants allowed qualitative modulation of CD4⁺ T‐cell behavior: poly I:C induced a strong IL‐12‐independent Th1 response, whereas curdlan led to the priming of Th17 cells. Thus, antigen targeting to DNGR‐1 is a versatile approach for inducing functionally distinct CD4⁺ T‐cell responses. Given the restricted pattern of expression of DNGR‐1 across species, this strategy could prove useful for developing immunotherapy protocols in humans.     

Interleukin‐10‐secreting T cells define a suppressive subset within the HIV‐1‐specific T‐cell population

Recent studies have indicated that Treg contribute to the HIV type 1 (HIV‐1)‐related immune pathogenesis. However, it is not clear whether T cells with suppressive properties reside within the HIV‐1‐specific T‐cell population. Here, PBMC from HIV‐1‐infected individuals were stimulated with a 15‐mer Gag peptide pool, and HIV‐1‐specific T cells were enriched by virtue of their secretion of IL‐10 or IFN‐γ using immunomagnetic cell‐sorting. Neither the IL‐10‐secreting cells nor the IFN‐γ‐secreting cells expressed the Treg marker FOXP3, yet the IL‐10‐secreting cells potently suppressed anti‐CD3/CD28‐induced CD4⁺ as well as CD8⁺ T‐cell proliferative responses. As shown by intracellular cytokine staining, IL‐10‐ and IFN‐γ‐producing T cells represent distinct subsets of the HIV‐1‐specific T cells. Our data collectively suggest that functionally defined HIV‐1‐specific T‐cell subsets harbor potent immunoregulatory properties that may contribute to HIV‐1‐associated T‐cell dysfunction.     

Human papillomavirus 16‐specific T cell responses in classic HPV‐related vulvar intra‐epithelial neoplasia. Determination of strongly immunogenic regions from E6 and E7 proteins

Cell‐mediated immunity directed against human papillomavirus 16 (HPV‐16) antigens was studied in 16 patients affected with classic vulvar intra‐epithelial neoplasia (VIN), also known as bowenoid papulosis (BP). Ten patients had blood lymphocyte proliferative T cell responses directed against E6/2 (14–34) and/or E6/4 (45–68) peptides, which were identified in the present study as immunodominant among HPV‐16 E6 and E7 large peptides. Ex vivo enzyme‐linked immunospot–interferon (IFN)‐γ assay was positive in three patients who had proliferative responses. Twelve months later, proliferative T cell responses remained detectable in only six women and the immunodominant antigens remained the E6/2 (14–34) and E6/4 (45–68) peptides. The latter large fragments of peptides contained many epitopes able to bind to at least seven human leucocyte antigen (HLA) class I molecules and were strong binders to seven HLA‐DR class II molecules. In order to build a therapeutic anti‐HPV‐16 vaccine, E6/2 (14–34) and E6/4 (45–68) fragments thus appear to be good candidates to increase HPV‐specific effector T lymphocyte responses and clear classic VIN (BP) disease lesions.     

Artificial human antigen‐presenting cells are superior to dendritic cells at inducing cytotoxic T‐cell responses

Peptide recognition through the MHC class I molecule by cytotoxic T lymphocytes (CTLs) leads to the killing of cancer cells. A potential challenge for T‐cell immunotherapy is that dendritic cells (DCs) are exposed to the MHC class I–peptide complex for an insufficient amount of time. To improve tumour antigen presentation to T cells and thereby initiate a more effective T‐cell response, we generated artificial antigen‐presenting cells (aAPCs) by incubating human immature DCs (imDCs) with poly(lactic‐co‐glycolic) acid nanoparticles (PLGA‐NPs) encapsulating tumour antigenic peptides, followed by maturation with lipopolysaccharide. Tumour antigen‐specific CTLs were then induced using either peptide‐loaded mature DCs (mDCs) or aAPCs, and their activities were analysed using both ELISpot and cytotoxicity assays. We found that the aAPCs induced significantly stronger tumour antigen‐specific CTL responses than the controls, which included both mDCs and aAPCs loaded with empty nanoparticles. Moreover, frozen CTLs that were generated by exposure to aAPCs retained the capability to eradicate HLA‐A2‐positive tumour antigen‐bearing cancer cells. These results indicated that aAPCs are superior to DCs when inducing the CTL response because the former are capable of continuously presenting tumour antigens to T cells in a sustained manner. The development of aAPCs with PLGA‐NPs encapsulating tumour antigenic peptides is a promising approach for the generation of effective CTL responses in vitro and warrants further assessments in clinical trials.     

Screening for fetal and neonatal alloimmune thrombocytopenia: is it possible and what are the potential outcomes?

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) is a rare but potentially very serious bleeding condition affecting the fetus/newborn. The vast majority (>80%) of FNAIT cases among Caucasians, and the most serious one, are caused by maternal antibodies to the human platelet antigen 1a (HPA‐1a). The clinical consequences of FNAIT span a continuum from no symptoms to intracranial haemorrhage (ICH) and intrauterine death. The incidence of FNAIT‐associated ICH has been estimated to be around 1 in 10 000 newborns. For many reasons, FNAIT has not been considered for prophylactic efforts similar to those that have been used for the prevention of HDFN, caused by antibodies against RhD: first, HPA‐1a typing kits are not well suited to the workflow in immunohaematology laboratories; secondly, the costs associated with HPA‐1a typing of all pregnant women have been a hindrance; thirdly, there is no international consensus regarding the optimal antenatal management of HPA‐1a‐immunized women identified in a screening programme; fourthly, it has generally been believed that immunization against HPA‐1a mostly takes place during the first incompatible pregnancy, and finally, there is yet no medicinal product available that can prevent HPA‐1a‐immunization in the same way as anti‐D can prevent RhD immunization. Implementation of HPA‐1a typing of all pregnant women has been discussed by the health authorities in several European countries. The abovementioned five hindrances for FNAIT screening and the Wilson and Jungner criteria for screening programmes will be discussed in the light of recent advances within FNAIT research.     

The Tat‐conjugated N‐terminal region of mucin antigen 1 (MUC1) induces protective immunity against MUC1‐expressing tumours

Mucin antigen 1 (MUC1) is overexpressed on various human adenocarcinomas and haematological malignancies and has long been used as a target antigen for cancer immunotherapy. Most of the preclinical and clinical studies using MUC1 have used the tandem repeat region of MUC1, which could be presented by only a limited set of major histocompatibility complex haplotypes. Here, we evaluated N‐terminal region (2–147 amino acids) of MUC1 (MUC1‐N) for dendritic cell (DC)‐based cancer immunotherapy. We used Esherichia coli‐derived MUC1‐N that was fused to the protein transduction domain of human immunodeficiency virus Tat protein for three reasons. First, mature DCs do not phagocytose soluble protein antigens. Secondly, tumour cells express underglycosylated MUC1, which can generate epitopes repertoire that differs from normal cells, which express hyperglycosylated MUC1. Finally, aberrantly glycosylated MUC1 has been known to impair DC function. In our study, Tat‐MUC1‐N‐loaded DCs induced type 1 T cell responses as well as cytotoxic T lymphocytes efficiently. Furthermore, they could break tolerance in the transgenic breast tumour mouse model, where MUC1‐positive breast cancers grow spontaneously. Compared with DCs pulsed with unconjugated MUC1‐N, DCs loaded with Tat‐conjugated MUC1‐N could delay tumour growth more effectively in the transgenic tumour model as well as in the tumour injection model. These results suggest that the recombinant N‐terminal part of MUC1, which may provide a diverse epitope repertoire, could be utilized as an effective tumour antigen for DC‐based cancer immunotherapy.     

Dimorphic HLA‐B signal peptides differentially influence HLA‐E‐ and natural killer cell‐mediated cytolysis of HIV‐1‐infected target cells

As a mechanism of self‐protection, signal peptides cleaved from human leukocyte antigen (HLA) class I products bind to HLA‐E before the complex interacts with the natural killer (NK) cell receptor CD94/NKG2A to inhibit NK‐mediated cell lysis. Two types of the signal peptides differ in their position 2 (P2) anchor residue, with P2‐methionine (P2‐M) having higher HLA‐E binding affinity than P2‐threonine (P2‐T). All HLA‐A and HLA‐C molecules carry P2‐M, whereas HLA‐B products have either P2‐M or P2‐T. Epidemiological evidence suggests that P2‐M is unfavourable in the context of HIV‐1 infection, being associated with accelerated acquisition of HIV‐1 infection in two African cohorts. To begin elucidating the functional mechanism, we studied NK‐mediated killing of CD4⁺ T cells and monocyte‐derived macrophages infected with two laboratory‐adapted HIV‐1 strains and two transmitted/founder (T/F) viruses. In the presence of target cells derived from individuals with the three HLA‐B P2 genotypes (M/M, M/T and T/T), NK‐mediated cytolysis was elevated consistently for P2‐T in a dose‐dependent manner for all cell and virus combinations tested (P = 0·008–0·03). Treatment of target cells with an anti‐HLA‐E monoclonal antibody restored NK‐mediated cytolysis of cells expressing P2‐M. Observations on cell lysis were also substantiated by measurements of HIV‐1 p24 antigen in the culture supernatants. Overall, our experiments indicate that the anti‐HIV‐1 function mediated by NK cells is compromised by P2‐M, corroborating the association of HLA‐B genotype encoding P2‐M with accelerated HIV‐1 acquisition.     

Influence of PD‐L1 cross‐linking on cell death in PD‐L1‐expressing cell lines and bovine lymphocytes

Programmed death‐ligand 1 (PD‐L1) blockade is accepted as a novel strategy for the reactivation of exhausted T cells that express programmed death‐1 (PD‐1). However, the mechanism of PD‐L1‐mediated inhibitory signalling after PD‐L1 cross‐linking by anti‐PD‐L1 monoclonal antibody (mAb) or PD‐1–immunogloblin fusion protein (PD‐1‐Ig) is still unknown, although it may induce cell death of PD‐L1⁺ cells required for regular immune reactions. In this study, PD‐1‐Ig or anti‐PD‐L1 mAb treatment was tested in cell lines that expressed PD‐L1 and bovine lymphocytes to investigate whether the treatment induces immune reactivation or PD‐L1‐mediated cell death. PD‐L1 cross‐linking by PD‐1‐Ig or anti‐PD‐L1 mAb primarily increased the number of dead cells in PD‐L1^high cells, but not in PD‐L1^low cells; these cells were prepared from Cos‐7 cells in which bovine PD‐L1 expression was induced by transfection. The PD‐L1‐mediated cell death also occurred in Cos‐7 and HeLa cells transfected with vectors only encoding the extracellular region of PD‐L1. In bovine lymphocytes, the anti‐PD‐L1 mAb treatment up‐regulated interferon‐γ (IFN‐γ) production, whereas PD‐1‐Ig treatment decreased this cytokine production and cell proliferation. The IFN‐γ production in B‐cell‐depleted peripheral blood mononuclear cells was not reduced by PD‐1‐Ig treatment and the percentages of dead cells in PD‐L1⁺ B cells were increased by PD‐1‐Ig treatment, indicating that PD‐1‐Ig‐induced immunosuppression in bovine lymphocytes could be caused by PD‐L1‐mediated B‐cell death. This study provides novel information for the understanding of signalling through PD‐L1.     

Mycobacterial antigen‐induced T helper type 1 (Th1) and Th2 reactivity of peripheral blood mononuclear cells from diabetic and non‐diabetic tuberculosis patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)‐vaccinated healthy subjects

Patients with diabetes mellitus are more susceptible to tuberculosis (TB), and the clinical conditions of diabetic TB patients deteriorate faster than non‐diabetic TB patients, but the immunological basis for this phenomenon is not understood clearly. Given the role of cell‐mediated immunity (CMI) in providing protection against TB, we investigated whether CMI responses in diabetic TB patients are compromised. Peripheral blood mononuclear cells (PBMC) obtained from diabetic TB patients, non‐diabetic TB patients and Mycobacterium bovis bacilli Calmette–Guérin (BCG)‐vaccinated healthy subjects were cultured in the presence of complex mycobacterial antigens and pools of M. tuberculosis regions of difference (RD)1, RD4, RD6 and RD10 peptides. The PBMC were assessed for antigen‐induced cell proliferation and secretion of T helper 1 (Th1) [interferon (IFN)‐γ, interleukin (IL)‐2, tumour necrosis factor (TNF)‐β], and Th2 (IL‐4, IL‐5, IL‐10) cytokines as CMI parameters. All the complex mycobacterial antigens and RD1_pool stimulated strong proliferation of PBMC of all groups, except moderate responses to RD1_pool in healthy subjects. In response to complex mycobacterial antigens, both IFN‐γ and TNF‐β were secreted by PBMC of all groups whereas diabetic TB patients secreted IL‐10 with concentrations higher than the other two groups. Furthermore, in response to RD peptides, IFN‐γ and IL‐10 were secreted by PBMC of diabetic TB patients only. The analyses of data in relation to relative cytokine concentrations showed that diabetic TB patients had lower Th1 : Th2 cytokines ratios, and a higher Th2 bias. The results demonstrate a shift towards Th2 bias in diabetic TB patients which may explain, at least in part, a faster deterioration in their clinical conditions.     

Analysis of nucleophosmin–anaplastic lymphoma kinase (NPM‐ALK)‐reactive CD8+ T cell responses in children with NPM‐ALK+ anaplastic large cell lymphoma

Cellular immune responses against the oncoantigen anaplastic lymphoma kinase (ALK) in patients with ALK‐positive anaplastic large cell lymphoma (ALCL) have been detected using peptide‐based approaches in individuals preselected for human leucocyte antigen (HLA)‐A*02:01. In this study, we aimed to evaluate nucleophosmin (NPM)‐ALK‐specific CD8⁺ T cell responses in ALCL patients ensuring endogenous peptide processing of ALK antigens and avoiding HLA preselection. We also examined the HLA class I restriction of ALK‐specific CD8⁺ T cells. Autologous dendritic cells (DCs) transfected with in‐vitro‐transcribed RNA (IVT‐RNA) encoding NPM–ALK were used as antigen‐presenting cells for T cell stimulation. Responder T lymphocytes were tested in interferon‐gamma enzyme‐linked immunospot (ELISPOT) assays with NPM–ALK‐transfected autologous DCs as well as CV‐1 in Origin with SV40 genes (COS‐7) cells co‐transfected with genes encoding the patients’ HLA class I alleles and with NPM–ALK encoding cDNA to verify responses and define the HLA restrictions of specific T cell responses. NPM–ALK‐specific CD8⁺ T cell responses were detected in three of five ALK‐positive ALCL patients tested between 1 and 13 years after diagnosis. The three patients had also maintained anti‐ALK antibody responses. No reactivity was detected in samples from five healthy donors. The NPM–ALK‐specific CD8⁺ T cell responses were restricted by HLA‐C‐alleles (C*06:02 and C*12:02) in all three cases. This approach allowed for the detection of NPM–ALK‐reactive T cells, irrespective of the individual HLA status, up to 9 years after ALCL diagnosis.     

Immunodominant T‐cell epitopes of hnRNP‐A2 associated with disease activity in patients with rheumatoid arthritis

The heterogeneous nuclear ribonucleoprotein A2 (hnRNP‐A2) has been described as an important autoantigen in rheumatoid arthritis (RA) since it is targeted by autoantibodies, autoreactive T cells, and is aberrantly expressed in synovial cells in patients. To identify hnRNP‐A2‐specific T‐cell epitopes possibly associated with pathogenicity, we used an innovative approach. We first scanned 280 overlapping hnRNP‐A2 peptides for binding to the RA‐associated class II molecules HLA‐DR4 and HLA‐DR1, leading to a comprehensive selection of binders. The selected peptides were tested in IFN‐γ‐specific ELISPOT assay: PBMC from 18% of RA patients showed a significant IFN‐γ response to hnRNP‐A2 peptides, 15% to the overlapping sequences 117–133 and/or 120–133, whereas PBMC from healthy individuals tested negative. We measured proliferative responses to these two peptides in another cohort of patients with RA or osteoarthritis: positive responses were found in 28% of RA, but also in 11% of osteoarthritis patients and these responses could be blocked by anti‐MHC class II Ab. Remarkably, the presence of 117/120–133‐specific T cells was significantly associated with active disease in RA patients, and bone erosion appeared to be more common in T‐cell positive patients. These data suggest involvement of hnRNP‐A2 specific cellular autoimmune responses in RA pathogenesis.     

Elevated and cross‐responsive CD1a‐reactive T cells in bee and wasp venom allergic individuals

The role of CD1a‐reactive T cells in human allergic disease is unknown. We have previously shown that circulating CD1a‐reactive T cells recognize neolipid antigens generated by bee and wasp venom phospholipase, and here tested the hypothesis that venom‐responsive CD1a‐reactive T cells associate with venom allergy. Circulating T cells from bee and wasp venom allergic individuals, before and during immunotherapy, were exposed to CD1a‐transfected K562 cells in the presence of wasp or bee venom. T‐cell response was evaluated based on IFNγ, GM‐CSF, and IL‐13 cytokine production. Venom allergic individuals showed significantly higher frequencies of IFN‐γ, GM‐CSF, and IL‐13 producing CD1a‐reactive T cells responsive to venom and venom‐derived phospholipase than healthy individuals. Venom‐responsive CD1a‐reactive T cells were cross‐responsive between wasp and bee suggesting shared pathways of allergenicity. Frequencies of CD1a‐reactive T cells were initially induced during subcutaneous immunotherapy, peaking by weeks 5, but then reduced despite escalation of antigen dose. Our current understanding of venom allergy and immunotherapy is largely based on peptide and protein‐specific T cell and antibody responses. Here, we show that lipid antigens and CD1a‐reactive T cells associate with the allergic response. These data have implications for mechanisms of allergy and approaches to immunotherapy.     

Genetic variants of immunoglobulin γ and κ chains influence humoral immunity to the cancer‐testis antigen XAGE‐1b (GAGED2a) in patients with non‐small cell lung cancer

GM (γ marker) allotypes, genetic variants of immunoglobulin γ chains, have been reported to be associated strongly with susceptibility to lung cancer, but the mechanism(s) underlying this association is not known. One mechanism could involve their contribution to humoral immunity to lung tumour‐associated antigens. In this study, we aimed to determine whether particular GM and KM (κ marker) allotypes were associated with antibody responsiveness to XAGE‐1b, a highly immunogenic lung tumour‐associated cancer‐testis antigen. Sera from 89 patients with non‐small cell lung cancer (NSCLC) were allotyped for eight GM and two KM determinants and characterized for antibodies to a synthetic XAGE‐1b protein. The distribution of various GM phenotypes was significantly different between XAGE‐1b antibody‐positive and ‐negative patients (P = 0·023), as well as in the subgroup of XAGE‐1b antigen‐positive advanced NSCLC (P = 0·007). None of the patients with the GM 1,17 21 phenotype was positive for the XAGE‐1b antibody. In patients with antigen‐positive advanced disease, the prevalence of GM 1,2,17 21 was significantly higher in the antibody‐positive group than in those who lacked the XAGE‐1b antibody (P = 0·026). This phenotype also interacted with a particular KM phenotype: subjects with GM 1,2,17 21 and KM 3,3 phenotypes were almost four times (odds ratio = 3·8) as likely to be positive for the XAGE‐1b antibody as the subjects who lacked these phenotypes. This is the first report presenting evidence for the involvement of immunoglobulin allotypes in immunity to a cancer‐testis antigen, which has important implications for XAGE‐1b‐based immunotherapeutic interventions in lung adenocarcinoma.     

CD1b‐mycolic acid tetramers demonstrate T‐cell fine specificity for mycobacterial lipid tails

Mycobacterium tuberculosis synthesizes a thick cell wall comprised of mycolic acids (MA), which are foreign antigens for human T cells. T‐cell clones from multiple donors were used to determine the fine specificity of MA recognition by human αβ T cells. Most CD1‐presented lipid antigens contain large hydrophilic head groups comprised of carbohydrates or peptides that dominate patterns of T‐cell specificity. MA diverges from the consensus antigen motif in that it lacks a head group. Using multiple forms of natural and synthetic MA and MA‐specific T‐cells with different T‐cell receptors, we found that, unlike antigens with larger head groups, lipid length strongly controlled T‐cell responses to MA. In addition, the three forms of MA that naturally occur in M. tuberculosis that differ in modifications on the lipid tail, differ in their potency for activating MA‐specific T‐cell clones. Thus, naturally occurring MA forms should be considered as separate, partly cross‐reactive antigens. Two of the three forms of MA could be loaded onto human CD1b proteins, creating working CD1b‐MA tetramers. The creation of CD1b‐MA tetramers represents a new tool for future studies that track the effector functions and kinetics of MA‐specific T‐cells ex vivo.     

Analysis of the functional WT1‐specific T‐cell repertoire in healthy donors reveals a discrepancy between CD4+ and CD8+ memory formation

The Wilms’ tumour‐1 (WT1) protein is considered a prime target for cancer immunotherapy based on its presumptive immunogenicity and widespread expression across a variety of malignancies. However, little is known about the naturally occurring WT1‐specific T‐cell repertoire because self‐derived antigens typically elicit low frequency responses that challenge the sensitivity limits of current detection techniques. In this study, we used highly efficient cell enrichment procedures based on CD137, CD154, and pHLA class I tetramer staining to conduct a detailed analysis of WT1‐specific T cells from the peripheral blood. Remarkably, we detected WT1‐specific CD4⁺ and CD8⁺ T‐cell populations in the vast majority of healthy individuals. Memory responses specific for WT1 were commonly present in the CD4⁺ T‐cell compartment, whereas WT1‐specific CD8⁺ T cells almost universally displayed a naive phenotype. Moreover, memory CD4⁺ and naive CD8⁺ T cells with specificity for WT1 were found to coexist in some individuals. Collectively, these findings suggest a natural discrepancy between the CD4⁺ and CD8⁺ T‐cell lineages with respect to memory formation in response to a self‐derived antigen. Nonetheless, WT1‐specific T cells from both lineages were readily activated ex vivo and expanded in vitro, supporting the use of strategies designed to exploit this expansive reservoir of self‐reactive T cells for immunotherapeutic purposes.