J. Anholts

Loss or downregulation of HLA class I expression at the allelic level in acute leukemia is infrequent but functionally relevant, and can be restored by interferon.

Human leukocyte antigen (HLA) class I expression at the allelic level was analyzed in 397 acute myeloid leukemia (AML) and 186 acute lymphoid leukemia (ALL) using a complement-dependent cytotoxicity assay. Impaired recognition possibly due to HLA downregulation was observed in 2% of the patients with AML and ALL in complete remission, and in 8%-15% in the groups with blasts. In 15 instances of diminished cytotoxicity, leukemic cells and control PHA blasts from the same patients were further analyzed using flow cytometry. In 4/6 ALL and 4/9 AML patients HLA downregulation or complete loss (2 patients) of cell surface expression could be confirmed. No genomic abnormalities were observed. In addition, 12 AML and 13 ALL patients were tested during relapse using flow cytometry. In 1/12 AML patients and 1/13 ALL patients allelic downregulation of cell surface expression was found. In two patients tested, downregulation or loss of cell surface expression of HLA class I antigens corresponded with impaired T cell mediated lysis by HLA restricted cytotoxic T lymphocyte.Treatment of the cells with alpha- or gamma-interferon could restore HLA class I expression and T-cell recognition. In conclusion, downregulation of cell surface expression of HLA class I expression at the allelic level in AML and ALL is infrequent but functionally relevant. HLA downregulation was reversible and T-cell recognition could be restored by alpha- or gamma-interferon.

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.

Polyclonal B cell activation for accurate analysis of pre-existing antigen-specific memory B cells

The enzyme‐linked immunospot (ELISPOT) assay is a widely used tool for enumeration of antigen‐specific memory B cells in several disciplines, such as vaccination, cancer immunotherapy and transplantation. For the accurate estimation of antigen‐specific memory B cell frequencies, a well‐defined B cell activation protocol is pivotal. In this study, we aimed to characterize a polyclonal B cell activation protocol to facilitate optimal monitoring of antigen‐specific memory B cell frequencies. Total, naive and memory B cells were activated polyclonally with an α‐CD40 monoclonal antibody, cytosine–phosphate–guanine (CPG) oligodeoxynucleotide (ODN) 2006, interleukin (IL)‐2, IL‐10 and IL‐21. Polyclonal activation of B cells resulted in equal cell death ratios in naive and memory B cells. When tested in an antigen‐specific system, immunoglobulin (Ig)G spots were detected only in the memory fraction. There was no change in B cell polyclonality due to in‐vitro activation. Our data show that the current polyclonal activation protocol may be used reliably to estimate the frequency of memory B cells in ELISPOT assays.

Increased metallothionein expression reflects steroid resistance in renal allograft recipients.

Steroid‐refractory acute rejection is a risk factor for inferior renal allograft outcome. We aimed to gain insight into the mechanisms underlying steroid resistance by identifying novel molecular markers of steroid‐refractory acute rejection. Eighty‐three kidney transplant recipients (1995–2005), who were treated with methylprednisolone during a first acute rejection episode, were included in this study. Gene expression patterns were investigated in a discovery cohort of 36 acute rejection biopsies, and verified in a validation cohort of 47 acute rejection biopsies. In the discovery set, expression of metallothioneins (MT) was significantly (p < 0.000001) associated with decreased response to steroid treatment. Multivariate analysis resulted in a predictive model containing MT‐1 as an independent covariate (AUC = 0.88, p < 0.0000001). In the validation set, MT‐1 expression was also significantly associated with steroid resistance (p = 0.029). Metallothionein expression was detected in macrophages and tubular epithelial cells. Parallel to the findings in patients, in vitro experiments of peripheral blood mononuclear cells from 11 donors showed that nonresponse to methylprednisolone treatment is related to highly elevated MT levels. High expression of metallothioneins in renal allografts is associated with resistance to steroid treatment. Metallothioneins regulate intracellular concentrations of zinc, through which they may diminish the zinc‐requiring anti‐inflammatory effect of the glucocorticoid receptor.

Beneficial Immune Effects of Myeloid-Related Proteins in Kidney Transplant Rejection.

Acute rejection is a risk factor for inferior long‐term kidney transplant survival. Although T cell immunity is considered the main effector in clinical acute rejection, the role of myeloid cells is less clear. Expression of S100 calcium‐binding protein A8 (S100A8) and S100A9 was evaluated in 303 biopsies before and after transplantation from 190 patients. In two independent cohorts of patients with acute rejection (n = 98 and n = 11; mostly cellular rejections), high expression of S100 calcium‐binding protein A8 (S100A8) and A9 (S100A9) was related to improved graft outcome. Mechanisms of action of the S100 molecules were investigated. In the graft and peripheral blood cells, S100A8 and S100A9 expression correlated with myeloid‐derived suppressor markers. In line with this finding, recombinant S100A8 and S100A9 proteins inhibited maturation and the allogeneic T cell stimulatory capacity of dendritic cells. S100A9 enhanced the production of reactive oxygen species by macrophages, which suppressed T cell activity at low concentrations in the form of hydrogen peroxide. Intragraft S100A8 and S100A9 expression linked to reduced expression of T cell immunity and tissue injury markers and higher expression of immune regulatory molecules. This study sheds new light on the importance of myeloid cell subsets in directing the outcome of T cell–mediated acute rejection.

Allo‐HLA Cross‐Reactivities of Cytomegalovirus‐, Influenza‐, and Varicella Zoster Virus–Specific Memory T Cells Are Shared by Different Healthy Individuals

Virus‐specific T cells can recognize allogeneic HLA (allo‐HLA) through TCR cross‐reactivity. The allospecificity often differs by individual (private cross‐reactivity) but also can be shared by multiple individuals (public cross‐reactivity); however, only a few examples of the latter have been described. Because these could facilitate alloreactivity prediction in transplantation, we aimed to identify novel public cross‐reactivities of human virus‐specific CD8+ T cells directed against allo‐HLA by assessing their reactivity in mixed‐lymphocyte reactions. Further characterization was done by studying TCR usage with primer‐based DNA sequencing, cytokine production with ELISAs, and cytotoxicity with 51chromium‐release assays. We identified three novel public allo‐HLA cross‐reactivities of human virus‐specific CD8+ T cells. CMV B35/IPS CD8+ T cells cross‐reacted with HLA‐B51 and/or HLA‐B58/B57 (23% of tetramer‐positive individuals), FLU A2/GIL (influenza IMP[58‐66] HLA‐A*02:01/GILGFVFTL) CD8+ T cells with HLA‐B38 (90% of tetramer‐positive individuals), and VZV A2/ALW (varicella zoster virus IE62[593‐601] HLA‐A*02:01/ALWALPHAA) CD8+ T cells with HLA‐B55 (two unrelated individuals). Cross‐reactivity was tested against different cell types including endothelial and epithelial cells. All cross‐reactive T cells expressed a memory phenotype, emphasizing the importance for transplantation. We conclude that public allo‐HLA cross‐reactivity of virus‐specific memory T cells is not uncommon and may create novel opportunities for alloreactivity prediction and risk estimation in transplantation.