Yoshiki Akatsuka

Some patients undergoing immune checkpoint blockade treatment develop immune-related adverse events (irAEs) affecting multiple organs, but whether pituitary and thyroid dysfunction are prone to co-occur is unclear. A total of 1,014 patients treated at Nagoya University Hospital were prospectively assessed for pituitary and thyroid function, and HLAs were analyzed in patients with pituitary and/or thyroid irAEs. Pituitary irAE and thyroid irAE developed in 68 and 128 patients, respectively. The incidence of thyroid irAE was significantly higher in patients who developed pituitary irAE compared to those who did not [21/68 (30.9%) vs. 107/946 (11.3%), p <0.001], and the difference remained significant with tumor type and therapeutic agents used as stratified factors (Cochran-Mantel-Haenszel test, p = 0.001). The frequencies of HLA-DRB1*15:01 (12.5% vs. 7.6%, p = 0.049) and DRB1*15:02 (16.9% vs 10.6%, p = 0.025) were significantly higher in patients with pituitary irAE compared to those in a database containing Japanese subjects. The frequency of the DRB1*15:01-associated haplotype (DRB1*15:01-DQB1*06:02-DPB1*02:01) was significantly higher in patients who developed both pituitary and thyroid irAEs compared to the control (14.3% vs. 3.1%, p = 0.002), and the DRB1*15:02-associated haplotype (DRB1*15:02-DQB1*06:01-DPB1*09:01) frequency was significantly higher in patients who developed pituitary irAE but not thyroid irAE compared to the control (18.1% vs. 8.9%, p = 0.006); both findings were confirmed in the validation cohort comprising 92 patients with pituitary irAE from seven hospitals. In conclusion, pituitary and thyroid irAEs are prone to co-occur and HLA-DR15-associated haplotypes are related to this co-occurrence.

OBJECTIVE: To report a case of bilateral reversible optic neuropathy as the first sign of Waldenström macroglobulinemia (WM). METHODS: Observational case report. RESULTS: A 52-year-old man had a sudden loss of vision in the left eye. Examinations revealed the presence of a serum monoclonal immunoglobulin (IgM kappa) in the serum. Even after a session of steroid pulse therapy, optic neuropathy became bilateral and then resolved almost completely after 4 months. The condition progressed to WM with multiorgan lesions years later. There was no evidence of optic neuropathy recurrence. The literature revealed two cases of monoclonal gammopathy (MG): a 64-year-old man with multiple myeloma (MM) with IgA lambda and a 51-year-old man with MM with IgG kappa. These cases have similar conditions: 1) visual reduction as an initial symptom of MG, 2) bilateral involvement, 3) no sign of central nervous system (CNS) infiltration shown by normal brain magnetic resonance images, and 4) recovery to a visual acuity of ≥1.0 bilaterally with no reoccurrence. The excessive Igs or B-cell hyperactivity may activate an autoimmune mechanism that reversibly interferes with the bilateral optic nerves. CONCLUSION: Bilateral optic neuropathy was the initial symptom of WM. There was no evidence of CNS infiltration; it recovered and then did not reoccur. The pathogenesis remained unknown, but two cases of MG were reported in the literature with remarkably similar conditions.

Relapsed leukemia after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a significant challenge, with the re-emergence of the primary disease being the most frequent cause of death. Human leukocyte antigen (HLA)-DPB1 mismatch occurs in approximately 70% of unrelated allo-HSCT cases, and targeting mismatched HLA-DPB1 is considered reasonable for treating relapsed leukemia following allo-HSCT if performed under proper conditions. In this study, we established several clones restricted to HLA-DPB1*02:01, -DPB1*04:02, and -DPB1*09:01 from three patients who underwent HLA-DPB1 mismatched allo-HSCT using donor-derived alloreactive T cells primed to mismatched HLA-DPB1 in the recipient's body after transplantation. A detailed analysis of the DPB1*09:01-restricted clone 2A9 showed reactivity against various leukemia cell lines and primary myeloid leukemia blasts, even with low HLA-DP expression. T cell receptor (TCR)-T cells derived from clone 2A9 retained the ability to trigger HLA-DPB1*09:01-restricted recognition and lysis of various leukemia cell lines in vitro. Our study demonstrated that the induction of mismatched HLA-DPB1 specific T cell clones from physiologically primed post-allo-HSCT alloreactive CD4+ T cells and the redirection of T cells with cloned TCR cDNA by gene transfer are feasible as techniques for future adoptive immunotherapy.

Negative immunofixation electrophoresis (IFE) of serum and/or urine is a diagnostic marker for determining a complete response (CR) after immunotherapy for multiple myeloma (MM). However, residual therapeutic antibodies such as elotuzumab (IgG-κ), can compromise IFE evaluation when the affected immunoglobulins belong to the same IgG-κ subclass. We thus sought to develop a simple and rapid method to treat patient serum before IFE to distinguish the residual elotuzumab. Serum samples from patients receiving elotuzumab were treated with a predetermined amount of soluble signaling lymphocyte activation molecule F7 (SLAMF7) protein and then subjected to conventional IFE testing. We tested our method in samples from 12 patients. The IgG-κ band in IFE disappeared or shifted after elotuzumab treatment in four patients with no bone marrow minimal residual disease and normalized free light chain, whereas seven patients with any sign of residual MM showed a remaining IgG-κ band after treatment. One-hour incubation of samples with 6-9 molar excess soluble SLAMF7 before IFE was sufficient to distinguish residual elotuzumab in 11 of 12 samples. This simple method does not require special reagents, can be performed in most clinical laboratories, and enables differentiation between patients with a CR and those requiring further treatment.

Minor histocompatibility antigens (mHAgs) in allogeneic hematopoietic stem cell transplantation are highly immunogenic as they are foreign antigens and cause polymorphism between donors and recipients. Adoptive cell therapy with mHAg-specific T cells may be an effective option for therapy against recurring hematological malignancies following transplantation. Genetically modified T cells with T cell receptors (TCRs) specific to mHAgs have been developed, but formation of mispaired chimeric TCRs between endogenous and exogenous TCR chains may compromise their function. An alternative approach is the development of chimeric antigen receptor (CAR)-T cells with TCR-like specificity whose CAR transmembrane and intracellular domains do not compete with endogenous TCR for CD3 complexes and transmit their own activation signals. However, it has been shown that the recognition of low-density antigens by high-affinity CAR-T cells has poor sensitivity and specificity. This mini review focuses on the potential for and limitations of TCR-like CAR-T cells in targeting human leukocyte antigen-bound peptide antigens, based on their recognition mechanisms and their application in targeting mHAgs.

[Background] Leukocytes that lack HLA class I alleles derived from hematopoietic stem progenitor cells (HSPCs) that undergo copy number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations are often detected in acquired aplastic anemia (AA) patients. The presence of HLA class I allele-lacking leukocytes provides compelling evidence that cytotoxic T lymphocytes (CTLs) are involved in the development of AA. Our recent study showed that, among several HLA-class I alleles that are likely to be lost as a result of 6pLOH, HLA-B*40:02 is the most frequently lost allele in AA. Therefore, HLA-B*4002 is thought to play a critical role in the autoantigen presentation by HSPCs to CTLs. We previously identified the T-cell receptor (TCR) sequences from bone marrow (BM) CD8+ T cells in two CsA-dependent AA patients possessing B4002-lacking leukocytes (Case 1, Espinoza et al, Blood Adv, 2018) and B5401-lacking leukocytes (Case 2, Elbadry et al, Haematologica, 2019) by single-cell T-cell receptor (TCR) sequencing. Identifying the TCRs specific to antigens presented by these HLA class I alleles should allow us to screen autoantigens in AA. [Method] We established B4002+ or B5401+ K562 cell lines expressing CD80 and CD137L for the screening of antigen-specific T cell responses. To identify ligands of the TCR, we transfected peripheral blood (PB) T cells with a retrovirus vector containing different TCR cDNA derived from BM T cells and examined their responses to B4002+CD80+CD137L+ or B5401+CD80+CD137L+ K562 cells. Specific responses of each TCR transfectant to K562 cells or iPSC-derived CD34+ cells were determined using an enzyme-linked immunosorbent assay for detecting IFN-γ. Deep TCR sequencing of a current PB sample taken from the same patients was performed to determine whether or not T cells with specific TCRs persisted after successful immunosuppressive therapy (IST). [Results] In Case 1, two TCR transfectants (TCR-K1 and TCR-K2 which were the third- and second-most frequent TCRs in the BM T cells, respectively) secreted greater IFN-γ levels (1730 pg/mL and 2157 pg/mL, respectively) in response to B4002+CD80+CD137L+ K562 cells than those secreted by the other six transfectants (710 to 1184 pg/mL, respectively). TCR-K1 and TCR-K2 did not respond to an A2402+ counterpart (Figure). Notably, deep TCR sequencing of a current PB sample taken from Case 1 nine years after BM sampling revealed the persistence of the TCR-K1 sequence, suggesting that TCR-K1 may be responsible for CsA dependency of this patient. Deep TCR sequencing of other three AA patients with B4002-lacking leukocytes revealed decreased diversity of the T cell repertoire in CD8+ T cells but failed to reveal the same TCR motifs as Case 1. In Case 2, two TCR transfectants (TCR-K3 and TCR-K4) showed a specific response to B5401+CD80+CD137L+ K562 cells. Furthermore, these 2 TCR transfectants secreted higher amounts of IFN-γ (1.7 and 2.0 folds for TCR-K3 and TCR-K4, respectively) in response to wild-type iPSC-derived CD34+ cells than to B5401(-) CD34+ cells. [Conclusions] Our results suggest that these TCR transfectants recognized some intrinsic antigens derived from K562 cells in a B4002 or B5401-restricted manner. These TCR transfectants are the ideal tools for screening libraries of cDNA expressed by B4002+ COS/293T cells to identify autoantigens in AA. Figure Disclosures Yoroidaka: Ono Pharmaceutical: Honoraria. Nakao:Takeda Pharmaceutical Company Limited: Honoraria; Bristol-Myers Squibb: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy; Chugai Pharmaceutical Co.,Ltd: Honoraria; Ono Pharmaceutical: Honoraria; Celgene: Honoraria; Kyowa Kirin: Honoraria; Novartis Pharma K.K: Honoraria.

Viral infection induces potent cellular immunity and activated intracellular signaling, which may dictate the driver events involved in immune escape and clonal selection of virus-associated cancers, including Epstein-Barr virus (EBV)-positive lymphomas. Here, we thoroughly interrogated PD-L1/PD-L2-involving somatic aberrations in 384 samples from various lymphoma subtypes using high-throughput sequencing, particularly focusing on virus-associated lymphomas. A high frequency of PD-L1/PD-L2-involving genetic aberrations was observed in EBV-positive lymphomas [33 (22%) of 148 cases], including extranodal NK/T-cell lymphoma (ENKTL, 23%), aggressive NK-cell leukemia (57%), systemic EBV-positive T-cell lymphoproliferative disorder (17%) as well as EBV-positive diffuse large B-cell lymphoma (DLBCL, 19%) and peripheral T-cell lymphoma-not otherwise specified (15%). Predominantly causing a truncation of the 3'-untranslated region, these alterations represented the most prevalent somatic lesions in ENKTL. By contrast, the frequency was much lower in EBV-negative lymphomas regardless of histology type [12 (5%) of 236 cases]. Besides PD-L1/PD-L2 alterations, EBV-positive DLBCL exhibited a genetic profile distinct from EBV-negative one, characterized by frequent TET2 and DNMT3A mutations and the paucity of CD79B, MYD88, CDKN2A, and FAS alterations. Our findings illustrate unique genetic features of EBV-associated lymphomas, also suggesting a potential role of detecting PD-L1/PD-L2-involving lesions for these lymphomas to be effectively targeted by immune checkpoint blockade.

BACKGROUND AND OBJECTIVES: Determination of the anti-A/-B titre pre- and post-transplantation is beneficial for treatment selection. Currently, the recommended method for antibody titration is the tube test (TT) assay. Dithiothreitol (DTT) is used for IgM antibody inactivation. Recently, a fully automated antibody titration assay using the column agglutination technique (CAT) was developed (auto-CAT). Our aim was to compare the auto-CAT and TT techniques for ABO antibody titration, to evaluate the effectiveness of DTT-treated plasma for use with auto-CAT and to define the cut-off value for antibody titration by auto-CAT. MATERIALS AND METHODS: We enrolled 30 healthy individuals, including 10 each for blood types A, B and O. We performed antibody titre measurement using the TT technique and auto-CAT simultaneously. Auto-CAT uses the bead column agglutination technology. RESULTS: With the auto-CAT cut-off value set to weak (w)+ with DTT treatment plasma, the concordance rate was 45%, and the weighted kappa value between TT and auto-CAT results was 0·994 in all subjects. Furthermore, there was a significant positive correlation between the anti-A/-B titre results obtained using the TT technique and auto-CAT in all blood types. Moreover, a positive bias (falsely elevated end-points due to agglomeration of A/B cells) was not observed in auto-CAT testing using DTT-treated plasma. CONCLUSION: Our results show that 1+ agglutination using the TT technique is equivalent to w+ agglutination obtained using auto-CAT. We recommend that DTT may be used with auto-CAT to measure antibody titres. Thus, we suggest that auto-CAT is useful for antibody titration in routine examination.

<title>Key Points</title> T cells expressing a CAR consisting of scFv #213 targeting WT1 peptide/HLA-A*2402 complex killed HLA-A*2402+ WT1+ tumor cell lines. The therapeutic efficacy of #213 scFv CAR-T cells was shown to be enhanced by DC vaccine in a murine xenograft model.

Hematopoietic stem cells (HSCs) that lack HLA-class I alleles as a result of copy-number neutral loss of heterozygosity of the short arm of chromosome 6 (6pLOH) or HLA allelic mutations often constitute hematopoiesis in patients with acquired aplastic anemia (AA), but the precise mechanisms underlying clonal hematopoiesis induced by these HLA-lacking (HLA-) HSCs remain unknown. To address this issue, we generated induced pluripotent stem cells (iPSCs) from an AA patient who possessed HLA-B4002-lacking (B4002-) leukocytes. Three different iPSC clones (wild-type [WT], 6pLOH+, and B*40:02-mutant) were established from the patient's monocytes. Three-week cultures of the iPSCs in the presence of various growth factors produced hematopoietic cells that make up 50% to 70% of the CD34+ cells of each phenotype. When 106 iPSC-derived CD34+ (iCD34+) cells with the 3 different genotypes were injected into the femoral bone of C57BL/6.Rag2 mice, 2.1% to 7.3% human multilineage CD45+ cells of each HLA phenotype were detected in the bone marrow, spleen, and peripheral blood of the mice at 9 to 12 weeks after the injection, with no significant difference in the human:mouse chimerism ratio among the 3 groups. Stimulation of the patient's CD8+ T cells with the WT iCD34+ cells generated a cytotoxic T lymphocyte (CTL) line capable of killing WT iCD34+ cells but not B4002- iCD34+ cells. These data suggest that B4002- iCD34+ cells show a repopulating ability similar to that of WT iCD34+ cells when autologous T cells are absent and CTL precursors capable of selectively killing WT HSCs are present in the patient's peripheral blood.

Selective graft-versus-tumor (GVT) reactivity with minimal risk of graft-versus-host disease (GVHD) following allogeneic stem cell transplantation is thought to be induced by targeting minor histocompatibility (H) antigens (Ags) expressed only on patients’ hematopoietic cells. Among HLA-A* 02:01 positive patients, minor H Ags such as HA-1 and HA-2 have been shown to be associated with anti-tumor responses with minimal GVHD and explored for application to adoptive immunotherapy. Because preparation of Ag-specific cytotoxic T cell clones (CTLs) or lines for adoptive immunotherapy is labor-intensive and time consuming, the genetic transfer of T-cell receptors (TCRs) directed toward target Ags into T lymphocytes has been used to efficiently generate anti-tumor T cells without the need for in vitro induction and expansion. Alternatively, T cells could be gene-modified with a chimeric antigen receptor (CAR) harnessing a single chain antibody moiety (scFv). The conventional CAR strategy has the limitation of only targeting cell surface Ags on target cells. One possible way to attain intracellular Ag targeting with a CAR is to generate a TCR-like monoclonal antibody (mAb) as a source of scFv. In this study, we sought to generate highly specific mAbs specific for HA-1H minor H Ag by immunizing mice with tetramerized recombinant HLA-A2 incorporating HA-1H minor H Ag peptides and β2-microglobulin (HA-1H/HLA-A2). We hypothesized that the use of HLA-A2 transgenic mice, which should be tolerant to human HLA-A2, would facilitate efficient induction of mAbs specific for peptides presented on HLA-A2. Phage libraries were generated from splenic B cells and screened by panning for clones reactive to plate-bound HA-1H/HLA-A2 in the presence of free MAGEA4/HLA-A2 for competition. Candidate scFv encoded by obtained phage clones were transformed to scFv tetrameric Ab form or introduced into T cells as CAR coupled to CD28 transmembrane and CD3ζ domains (CD28-ζ). A total of 144 clones were randomly selected from 8.1×108 clones that had been recovered after the third panning. Among 144 clones, 18 (12.5%) showed preferential binding to HA-1/HLA-A2, 137 showed similar binding to both pMHC complexes, and 7 showed reactivity to neither of them. One of 18 scFv Abs, clone #131, demonstrated high affinity (KD = 8.34nM) for the HA-1H/HLA-A2 complex. Primary human CD8 T cells transduced with #131 scFv-CD28-ζ were stained with HA-1H/HLA-A2 tetramers as strongly as a CTL clone, EH6, specific for endogenously HLA-A2- and HA-1H-positive cells. Unexpectedly, however, #131 scFv-CD28-ζ CAR-T cells required ∼100-fold higher Ag density when pulsed exogenously to exert cytotoxicity than did the cognate EH6-CTL. In addition, mAb blocking experiments demonstrated that #131 scFv-CD28-ζCAR-T cells were less sensitive to CD8 blockade when they were completely blocked with HA-1H/HLA-A2 tetramer. These data suggest that T cells with higher affinity antigen receptors than TCRs (average KD ranging between 1μM∼100μM) are less able to recognize low density peptide/MHC antigens as reported in the case of affinity-matured TCR or CAR, and that CD8+ CAR-T cells may not be necessarily CD8-dependent possibly due to failure to form complexes with CD3. <sec> <title>Disclosures:</title> No relevant conflicts of interest to declare. </sec>