Single-cell landscape analysis unravels molecular programming of the human B cell compartment in chronic GVHD.

Alloreactivity can drive autoimmune syndromes. After allogeneic hematopoietic stem cell transplantation (allo-HCT), chronic graft-versus-host disease (cGVHD), a B cell-associated autoimmune-like syndrome, commonly occurs. Because donor-derived B cells continually develop under selective pressure from host alloantigens, aberrant B cell receptor (BCR) activation and IgG production can emerge and contribute to cGVHD pathobiology. To better understand molecular programing of B cells in allo-HCT, we performed scRNA-Seq analysis on high numbers of purified B cells from patients. An unsupervised analysis revealed 10 clusters, distinguishable by signature genes for maturation, activation, and memory. Within the memory B cell compartment, we found striking transcriptional differences in allo-HCT patients compared with healthy or infected individuals, including potentially pathogenic atypical B cells (ABCs) that were expanded in active cGVHD. To identify intrinsic alterations in potentially pathological B cells, we interrogated all clusters for differentially expressed genes (DEGs) in active cGVHD versus patients who never had signs of immune tolerance loss (no cGVHD). Active cGVHD DEGs occurred in both naive and BCR-activated B cell clusters. Remarkably, some DEGs occurred across most clusters, suggesting common molecular programs that may promote B cell plasticity. Our study of human allo-HCT and cGVHD provides understanding of altered B cell memory during chronic alloantigen stimulation.

Antibodies targeting surface membrane antigens in patients with chronic graft-versus-host disease.

Chronic graft-versus-host disease (cGVHD) after allogeneic hematopoietic stem cell transplant reflects a complex immune response resulting in chronic damage to multiple tissues. Previous studies indicated that donor B cells and the antibodies they produce play an important role in the development of cGVHD. To understand the pathogenic role of antibodies in cGVHD, we focused our studies on posttransplant production of immunoglobulin G antibodies targeting cell surface antigens expressed in multiple cGVHD affected tissues, due to their potential functional impact on living cells in vivo. Using plate-bound cell membrane proteins as targets, we detected a significantly higher level of antibodies reactive with these membrane antigens in patients who developed cGVHD, compared with those who did not and healthy donors. Plasma-reactive antibody levels increased significantly prior to the clinical diagnosis of cGVHD and were reduced following cGVHD therapies including prednisone, interleukin-2, or extracorporeal photophoresis. Using cell-based immunoprecipitation with plasma from cGVHD patients and mass spectrometry, we identified 43 membrane proteins targeted by these antibodies. The presence of antibodies in cGVHD patients' plasma that specifically target 6 of these proteins was validated. Antibodies reactive with these 6 antigens were more frequently detected in patients with cGVHD compared with patients without cGVHD and healthy donors. These results indicate that antibodies that target membrane antigens of living cells frequently develop in cGVHD patients and further support a role for B cells and antibodies in the development of cGVHD.

Gene expression profiles of B-lineage adult acute lymphocytic leukemia reveal genetic patterns that identify lineage derivation and distinct mechanisms of transformation.

PURPOSE: To characterize gene expression signatures in acute lymphocytic leukemia (ALL) cells associated with known genotypic abnormalities in adult patients. EXPERIMENTAL DESIGN: Gene expression profiles from 128 adult patients with newly diagnosed ALL were characterized using high-density oligonucleotide microarrays. All patients were enrolled in the Italian GIMEMA multicenter clinical trial 0496 and samples had >90% leukemic cells. Uniform phenotypic, cytogenetic, and molecular data were also available for all cases. RESULTS: T-lineage ALL was characterized by a homogeneous gene expression pattern, whereas several subgroups of B-lineage ALL were evident. Within B-lineage ALL, distinct signatures were associated with ALL1/AF4 and E2A/PBX1 gene rearrangements. Expression profiles associated with ALL1/AF4 and E2A/PBX1 are similar in adults and children. BCR/ABL+ gene expression pattern was more heterogeneous and was most similar to ALL without known molecular rearrangements. We also identified a set of 83 genes that were highly expressed in leukemia blasts from patients without known molecular abnormalities who subsequently relapsed following therapy. Supervised analysis of kinase genes revealed a high-level FLT3 expression in a subset of cases without molecular rearrangements. Two other kinases (PRKCB1 and DDR1) were highly expressed in cases without molecular rearrangements, as well as in BCR/ABL-positive ALL. CONCLUSIONS: Genomic signatures are associated with phenotypically and molecularly well defined subgroups of adult ALL. Genomic profiling also identifies genes associated with poor outcome in cases without molecular aberrations and specific genes that may be new therapeutic targets in adult ALL.

Infusion of CD4+ donor lymphocytes induces the expansion of CD8+ donor T cells with cytolytic activity directed against recipient hematopoietic cells.

PURPOSE: Donor lymphocyte infusions (DLIs) provide effectivetherapy for patients with relapsed chronic myeloid leukemia after allogeneic bone marrow transplantation. Previous studies have suggested that depletion of CD8+ T cells from the infused donor lymphocytes can reduce the incidence of graft-versus-host disease associated with DLI without reducing antileukemia activity. In this situation however, the immune effector cells responsible for tumor rejection have not been identified. The goal of this study was to characterize these effector populations. EXPERIMENTAL DESIGN: We studied three representative patients with relapsed chronic myeloid leukemia who achieved complete molecular remission after receiving CD8+ T-cell-depleted DLI from HLA-identical sibling donors. Effector T cells were characterized in patient samples after in vitro stimulation and functional assessment. T-cell clones relevant to the immune response were then isolated and further characterized. RESULTS: Analysis of peripheral blood samples collected after DLI indicated the presence of a high frequency of circulating host-reactive cytolytic CD8+ T cells secreting IFN-gamma. These HLA class I-restricted CTLs were specific for recipient minor histocompatibility antigens (mHAs) because they did not recognize target cells of donor origin. One CTL clone was further expanded in vitro and shown to recognize a broadly expressed mHA presented by HLA-B5701. Using a molecular approach, we demonstrated that this clone was expanded in peripheral blood and marrow after DLI. It was not detected before DLI. CONCLUSIONS: These results indicate that CD4+ DLI elicits a potent allogeneic response mediated by mHA-specific CD8+ T cells.

Synergistic enhancement of cell-mediated immunity by interleukin-12 plus interleukin-2: basis for therapy of cutaneous T cell lymphoma.

Cutaneous T cell lymphoma is a clonally derived, skin-invasive malignancy of CD4+ T lymphocytes with the phenotype of mature helper T cells. Previous work has demonstrated that the Sézary form, or typically leukemic form of cutaneous T cell lymphoma, is characterized by prominent immunologic defects, including depressed cell-mediated immunity associated with marked defects in the production of interleukin-12 and other type 1 helper T cell cytokines. Recent clinical trials with recombinant human interleukin-12 for cutaneous T cell lymphoma have demonstrated that it is a potent therapeutic agent, which induces cytotoxic T cell responses. Nevertheless, a high rate of refractoriness to recombinant human interleukin-12 occurred in these studies that may be related to the downmodulation of interleukin-12 receptor expression by chronic interleukin-12 use. In an effort to enhance the overall response rate and to overcome the refractoriness to recombinant human interleukin-12 therapy, we studied the immunologic effects in vitro of adding interleukin-2 to interleukin-12 as a model to achieve these goals. We examined the stimulation of interferon-gamma production, natural killer cell activity and interleukin-12 receptor expression by T cells of cutaneous T cell lymphoma patients. The addition of interleukin-12 to cutaneous T cell lymphoma patient peripheral blood cells resulted in the production of interferon-gamma (mean = 7914 pg per ml +/- 2161, n = 15) as did interleukin-2 alone (mean = 7222 pg per ml +/- 2228, n = 15). Importantly, the addition of interleukin-2 to the interleukin-12 synergistically enhanced the levels of interferon-gamma produced (mean = 16 792 pg per ml +/- 2492 n = 15) (p <0.01). Similarly, addition of interleukin-2 to interleukin-12 synergistically enhanced both the natural killer cell activity of 15 cutaneous T cell lymphoma patients as well as T cell surface interleukin-12 receptor expression in comparison with the effects of interleukin-12 or interleukin-2 alone. Thus, interleukin-2 plus interleukin-12 unequivocally produces the synergistic enhancement of multiple parameters of cell-mediated immunity as well as upmodulating interleukin-12 receptor expression; this indicates that protocols combining these two potent immune enhancing cytokines may have added therapeutic benefit for cutaneous T cell lymphoma.