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Supplementary Data from Olfactory Receptor OR2H1 Is an Effective Target for CAR T Cells in Human Epithelial Tumors

Supplementary Figure from Olfactory Receptor OR2H1 Is an Effective Target for CAR T Cells in Human Epithelial Tumors

Recent studies suggest that B cells could play an important role in the tumor microenvironment. However, the role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we evaluated the role of coordinated humoral and cellular adaptive immune responses in endometrial cancer. Concomitant accumulation of T, B, and plasma cells at tumor beds predicted better survival. However, only B-cell markers corresponded with prolonged survival specifically in high-grade endometrioid type and serous tumors. Immune protection was associated with class-switched IgA and, to a lesser extent, IgG. Expressions of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA were superior predictors of outcome and correlated with defects in methyl-directed DNA mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drove activation of inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and endoplasmic reticulum stress pathways, while thwarting DNA repair mechanisms. Together, these findings suggest that coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer as well as the potential for effective immunotherapies.Significance:This study provides new insights into the crucial role of humoral immunity in human endometrial cancer, providing a rationale for designing novel immunotherapies against this prevalent malignancy.See related commentary by Osorio and Zamarin, p. 766

Supplementary Figure from IgA-Dominated Humoral Immune Responses Govern Patients' Outcome in Endometrial Cancer

Recent studies suggest that B cells could play an important role in the tumor microenvironment. However, the role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we evaluated the role of coordinated humoral and cellular adaptive immune responses in endometrial cancer. Concomitant accumulation of T, B, and plasma cells at tumor beds predicted better survival. However, only B-cell markers corresponded with prolonged survival specifically in high-grade endometrioid type and serous tumors. Immune protection was associated with class-switched IgA and, to a lesser extent, IgG. Expressions of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA were superior predictors of outcome and correlated with defects in methyl-directed DNA mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drove activation of inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and endoplasmic reticulum stress pathways, while thwarting DNA repair mechanisms. Together, these findings suggest that coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer as well as the potential for effective immunotherapies. Significance: This study provides new insights into the crucial role of humoral immunity in human endometrial cancer, providing a rationale for designing novel immunotherapies against this prevalent malignancy. See related commentary by Osorio and Zamarin, p. 766

Although chimeric antigen receptor (CAR)-expressing T cells have proven success in hematologic malignancies, their effectiveness in solid tumors has been largely unsuccessful thus far. We found that some olfactory receptors are expressed in a variety of solid tumors of different histologic subtypes, with a limited pattern of expression in normal tissues. Quantification of OR2H1 expression by qRT-PCR and Western blot analysis of 17 normal tissues, 82 ovarian cancers of various histologies, eight non–small cell lung cancers (NSCLCs), and 17 breast cancers demonstrated widespread OR2H1 expression in solid epithelial tumors with expression in normal human tissues limited to the testis. CAR T cells recognizing the extracellular domain of the olfactory receptor OR2H1 were generated with a targeting motif identified through the screening of a phage display library and demonstrated OR2H1-specific cytotoxic killing in vitro and in vivo, using tumor cells with spontaneous expression of variable OR2H1 levels. Importantly, recombinant OR2H1 IgG generated with the VH/VL sequences of the CAR construct specifically detected OR2H1 protein signal in 60 human lung cancers, 40 ovarian carcinomas, and 73 cholangiocarcinomas, at positivity rates comparable with mRNA expression and without OR2H1 staining in 58 normal tissues. CRISPR/Cas9-mediated ablation of OR2H1 confirmed targeting specificity of the CAR and the tumor-promoting role of OR2H1 in glucose metabolism. Therefore, T cells redirected against OR2H1-expressing tumor cells represent a promising therapy against a broad range of epithelial cancers, likely with an admissible toxicity profile.

Most ovarian cancers are infiltrated by prognostically relevant activated T cells1–3, yet exhibit low response rates to immune checkpoint inhibitors4. Memory B cell and plasma cell infiltrates have previously been associated with better outcomes in ovarian cancer5,6, but the nature and functional relevance of these responses are controversial. Here, using 3 independent cohorts that in total comprise 534 patients with high-grade serous ovarian cancer, we show that robust, protective humoral responses are dominated by the production of polyclonal IgA, which binds to polymeric IgA receptors that are universally expressed on ovarian cancer cells. Notably, tumour B-cell-derived IgA redirects myeloid cells against extracellular oncogenic drivers, which causes tumour cell death. In addition, IgA transcytosis through malignant epithelial cells elicits transcriptional changes that antagonize the RAS pathway and sensitize tumour cells to cytolytic killing by T cells, which also contributes to hindering malignant progression. Thus, tumour-antigen-specific and -antigen-independent IgA responses antagonize the growth of ovarian cancer by governing coordinated tumour cell, T cell and B cell responses. These findings provide a platform for identifying targets that are spontaneously recognized by intratumoural B-cell-derived antibodies, and suggest that immunotherapies that augment B cell responses may be more effective than approaches that focus on T cells, particularly for malignancies that are resistant to checkpoint inhibitors. In patients with high-grade serous ovarian cancer, robust and protective humoral responses are dominated by B-cell-derived polyclonal IgA that binds to polymeric IgA receptors that are universally expressed on ovarian cancer cells.

Despite repeated associations between T cell infiltration and outcome, human ovarian cancer remains poorly responsive to immunotherapy. We report that the hallmarks of tumor recognition in ovarian cancer-infiltrating T cells are primarily restricted to tissue-resident memory (TRM) cells. Single-cell RNA/TCR/ATAC sequencing of 83,454 CD3(+) CD8(+)CD103(+)CD69(+) TRM cells and immunohistochemistry of 122 high-grade serous ovarian cancers shows that only progenitor (TCF1(low)) tissue-resident T cells (TRMstem cells), but not recirculating TCF1(+) T cells, predict ovarian cancer outcome. TRMstem cells arise from transitional recirculating T cells, which depends on antigen affinity/persistence, resulting in oligoclonal, trogocytic, effector lymphocytes that eventually become exhausted. Therefore, ovarian cancer is indeed an immunogenic disease, but that depends on similar to 13% of CD8(+) tumor-infiltrating T cells (similar to 3% of CD8(+) clonotypes), which are primed against high-affinity antigens and maintain waves of effector TRM-like cells. Our results define the signature of relevant tumor-reactive T cells in human ovarian cancer, which could be applicable to other tumors with unideal mutational burden.

Gamma delta (γδ) T cells infiltrate most human tumors, but current immunotherapies fail to exploit their in situ major histocompatibility complex-independent tumoricidal potential. Activation of γδ T cells can be elicited by butyrophilin and butyrophilin-like molecules that are structurally similar to the immunosuppressive B7 family members, yet how they regulate and coordinate αβ and γδ T cell responses remains unknown. Here, we report that the butyrophilin BTN3A1 inhibits tumor-reactive αβ T cell receptor activation by preventing segregation of N-glycosylated CD45 from the immune synapse. Notably, CD277-specific antibodies elicit coordinated restoration of αβ T cell effector activity and BTN2A1-dependent γδ lymphocyte cytotoxicity against BTN3A1+ cancer cells, abrogating malignant progression. Targeting BTN3A1 therefore orchestrates cooperative killing of established tumors by αβ and γδ T cells and may present a treatment strategy for tumors resistant to existing immunotherapies.

e14507 Background: We hypothesized that tumor-infiltrating B cells in endometriosis and endometriosis-associated ovarian cancer can be used to identify novel, targetable antigen domains to inhibit the progression of ovarian carcinomas. We aimed to identify targets that are spontaneously recognized by B-cell-derived antibodies within ovarian clear cell carcinoma (CCC), endometrioid carcinoma (EC), and endometriosis and to determine the preclinical anti-cancer efficacy of a candidate antibody that recognizes the extracellular domain of an identified target. Methods: B cells from freshly dissociated CCC (n = 2), EC (n = 2), and endometriomas (n = 2) were isolated, activated, and EBV-immortalized. Antibodies were purified from B cell supernatants and used for screening in a proteome array. Targets were prioritized based on accessibility (transmembrane or secreted proteins), expression in endometriosis and cancer, and concurrent IgA and IgG responses. Tumor-promoting syndecan binding protein (SDCBP) was identified and SDCBP-reactive B cells were FACS-sorted and subjected to single cell B cell receptor sequencing to determine the variable heavy and light chain sequences of enriched clonotypes. We then generated a recombinant IgG4 antibody targeting SDCBP with the dominant VH/VL matching sequences. Immunoblots and qPCR were performed to assess SDCBP expression in ovarian cancer cell lines and tumor samples. In vivo studies compared anti-tumor potential of the α-SDCBP IgG4 with controls using immunodeficient mouse models of human CCC and high-grade serous ovarian carcinoma (HGSOC). Results: Nine accessible proteins were detected by both IgA and IgG in all samples, including SDCBP. SDCBP is expressed in ovarian cancer cell lines and tumor samples of multiple histologies, including CCC, EC, and HGSOC. Administration of α-SDCBP IgG4 in TOV21G (CCC) tumor-bearing mice significantly decreased tumor volume compared to non-antigen-specific irrelevant IgG4 (iIgG4, p = 0.002) and vehicle (0.001), and correspondingly trended toward decreased tumor weight compared to vehicle (p = 0.06). Likewise, administration of α-SDCBP IgG4 in OVCAR3 (HGSOC) tumor-bearing mice significantly decreased tumor volume compared to iIgG4 (p = 0.03) and trended toward decreased tumor weight (p = 0.06). Conclusions: An α-SDCBP IgG4 has demonstrated anti-tumor efficacy in SDCBP+ CCC and HGSOC, and SDCBP-targeted therapy for endometriosis and associated malignant conditions, as well as HGSOC, warrants further investigation.

Cutaneous T cell lymphoma (CTCL) has a poorly understood etiology and no known cure. Using conditional knockout mice, we found that ablation of the genomic organizer special AT-rich sequence–binding protein 1 (Satb1) caused malignant transformation of mature, skin-homing, Notch-activated CD4(+) and CD8(+) T cells into progressively fatal lymphoma. Mechanistically, Satb1 restrained Stat5 phosphorylation and the expression of skin-homing chemokine receptors in mature T cells. Notably, methyltransferase-dependent epigenetic repression of SATB1 was universally found in human Sézary syndrome, but not in other peripheral T cell malignancies. H3K27 and H3K9 trimethylation occluded the SATB1 promoter in Sézary cells, while inhibition of SUV39H1/2 methyltransferases (unlike EZH2 inhibition) restored protective SATB1 expression and selectively abrogated the growth of primary Sézary cells more effectively than romidepsin. Therefore, inhibition of methyltransferases that silence SATB1 could address an unmet need for patients with mycosis fungoides/Sézary syndrome, a set of incurable diseases.

Tertiary Lymphoid Structures (TLS) are commonly identified in human tumors with improved outcome, but how they are orchestrated remains elusive. We show that ablation of the genomic organizer SATB1 triggers the polarization of CD4+ T-cells into antigen-specific T Follicular Helper (TFH) cells. In contrast, SATB1 expression is required for conversion of CD4+ T-cells into induced regulatory T-cells (Treg), including T Follicular Regulatory (TFR) cells. Paradoxically, Tgf-β-driven Satb1 silencing causes Icos de-repression and prevents TFR differentiation, driving TFH polarization and increased isotype-switched B cell responses. Tumors in mice carrying Satb1-deficient CD4+ T-cells accumulate more tumor antigen-specific, Light/Cxcl13/Il-21-producing TFH cells, causing germinal center formation and spontaneous TLS assembly, which antagonizes tumor growth. Accordingly, TFH cell transfer induces large TLS within orthotopic ovarian tumors. Therefore, SATB1 repression in CD4+ T-cells licenses TFH cell differentiation while preventing Treg conversion, a central mechanism during TLS assembly that can be exploited to boost anti-tumor immunity.

Despite repeated associations between T-cell infiltration and patient outcome, human ovarian cancer remains poorly responsive to immunotherapy. We report that hallmarks of tumor recognition in ovarian cancer-infiltrating T-cells are primarily restricted to tissue-resident memory (TRM) cells. In mouse models we found that TRM T-cells were better than the re-circulating counterpart at controlling tumor growth. Single-cell RNA/TCR/ATAC sequencing of 83,454 CD3+CD8+CD103+CD69+ TRM cells and 24,175 CD3+CD8+CD103− re-circulating TILs showed that progenitor (TCF1low) tissue-resident memory T-cells (TRMstem cells) arise from transitional recirculating T-cells, which depends on antigen affinity/persistence, resulting in oligoclonal, trogocytic, effector lymphocytes. This effector population develops into proliferative lymphocytes that eventually become exhausted TRMs. Immunohistochemistry of 122 high-grade serous ovarian cancer tissues showed that only TRMstem cells, but not re-circulating TCF1+ T-cells, predict ovarian cancer outcome. Therefore, ovarian cancer is indeed an immunogenic disease that depends on ~13% of CD8+ tumor-infiltrating T-cells (~3% of CD8+ clonotypes), which are primed against high-affinity antigens and maintain waves of effector TRM cells. Support for Shared Resources was provided by Cancer Center Support Grant (CCSG) CA076292 to H. Lee Moffitt Cancer Center and by CCSG CA010815 to The Wistar Institute. This study was supported by grants from NIH (R01CA157664, R01CA124515, R01CA178687, R01CA211913 and U01CA232758 to JRCG; R01CA184185 and RO1CA262121 to PCR.)

The role of humoral responses in endometrial cancer remains insufficiently investigated. Using a cohort of 107 patients with different histological subtypes of endometrial carcinoma, we report that concomitant accumulation of T, B and plasma cells at tumor beds predicts better survival. However, only B cell markers predict survival specifically in high-grade endometrioid type and serous tumors. Accordingly, immune protection is associated with class-switched IgA and, to a lesser extent, IgG. Notably, expression of polymeric immunoglobulin receptor (pIgR) by tumor cells and its occupancy by IgA are superior predictors of outcome, and correlate with defects in methyl mismatch repair. Mechanistically, pIgR-dependent, antigen-independent IgA occupancy drives inflammatory pathways associated with IFN and TNF signaling in tumor cells, along with apoptotic and ER stress pathways, while thwarting DNA repair mechanisms. Therefore, coordinated humoral and cellular immune responses, characterized by IgA:pIgR interactions in tumor cells, determine the progression of human endometrial cancer, and therefore the potential for effective immunotherapies. Supported by grants from NIH (R01CA157664, R01CA124515, R01CA178687 and R01CA211913), and from Cancer Center Support Grant (CCSG) CA076292

The immune checkpoint receptor PD-1 on T follicular helper (Tfh) cells promotes Tfh:B cell interactions and appropriate positioning within tissues. Here, we examined the impact of regulation of PD-1 expression by the genomic organizer SATB1 on Tfh cell differentiation. Vaccination of CD4

Despite the importance of programmed cell death-1 (PD-1) in inhibiting T cell effector activity, the mechanisms regulating its expression remain poorly defined. We found that the chromatin organizer special AT-rich sequence-binding protein-1 (Satb1) restrains PD-1 expression induced upon T cell activation by recruiting a nucleosome remodeling deacetylase (NuRD) complex to Pdcd1 regulatory regions. Satb1 deficienct T cells exhibited a 40-fold increase in PD-1 expression. Tumor-derived transforming growth factor β (Tgf-β) decreased Satb1 expression through binding of Smad proteins to the Satb1 promoter. Smad proteins also competed with the Satb1-NuRD complex for binding to Pdcd1 enhancers, releasing Pdcd1 expression from Satb1-mediated repression, Satb1-deficient tumor-reactive T cells lost effector activity more rapidly than wild-type lymphocytes at tumor beds expressing PD-1 ligand (CD274), and these differences were abrogated by sustained CD274 blockade. Our findings suggest that Satb1 functions to prevent premature T cell exhaustion by regulating Pdcd1 expression upon T cell activation. Dysregulation of this pathway in tumor-infiltrating T cells results in diminished anti-tumor immunity.

In this study, we deploy a doxycycline-dependent suicide switch integrated in a tumor challenge model. With this experimental setup, we characterized the immunological consequences of cells dying by four distinct cell death stimuli in vivo. We observed that apoptotic cell death induced by expression of the truncated form of BH3 interacting-domain death agonist (tBid) and a constitutively active form of caspase 3 (revC3), respectively, showed higher immunogenicity than cell death induced by expression of the tuberculosis-necrotizing toxin (TNT). Our data indicate that the early release of ATP induces the silent clearance of dying cells, whereas the simultaneous presence of ‘find me' signals and danger-associated molecular patterns (DAMPs) promotes inflammatory reactions and increased immunogenicity. This proposed model is supported by findings showing that the production and release of high concentrations of IL-27 by bone-marrow-derived macrophages (BMDM) is limited to BMDM exposed to those forms of death that simultaneously released ATP and the DAMPs heat-shock protein 90 (HSP90) and high-mobility group box-1 protein (HMGB1). These results demonstrate that the tissue microenvironment generated by dying cells may determine the subsequent immune response.

Solid cancers progress from primordial lesions through complex interactions between tumor-promoting and anti-tumor immune cell types, ultimately leading to the orchestration of humoral and T cell adaptive immune responses, albeit in an immunosuppressive environment. B cells infiltrating most established tumors have been associated with a dual role: Some studies have associated antibodies produced by tumor-associated B cells with the promotion of regulatory activities on myeloid cells, and also with direct immunosuppression through the production of IL-10, IL-35 or TGF-β. In contrast, recent studies in multiple human malignancies identify B cell responses with delayed malignant progression and coordinated T cell protective responses. This includes the elusive role of Tertiary Lymphoid Structures identified in many human tumors, where the function of B cells remains unknown. Here, we discuss emerging data on the dual role of B cell responses in the pathophysiology of human cancer, providing a perspective on future directions and possible novel interventions to restore the coordinated action of both branches of the adaptive immune response, with the goal of maximizing immunotherapeutic effectiveness.

Cutaneous T cell lymphoma (CTCL) is a clinically unmet need. Using conditional knockout mice, we found that ablation of the genomic organizer Special AT rich sequence binding protein 1 (Satb1) induces a progressively fatal lymphoma characterized by mature, skin homing, Notch activated CD4 and CD8 T cells. Mechanistically, Satb1 restrains Stat5 phosphorylation and the expression of skin homing chemokine receptors in mature T cells. Notably, SUV39H1 and 2 methyltransferase dependent epigenetic repression of SATB1 is universally found in human Sezary Syndrome, but not other peripheral T cell malignancies. Accordingly, H3K27 and H3K9 trimethylation occlude the SATB1 promoter in Sezary cells. Inhibition of SUV39H1 and 2 methyltransferases with novel drugs, unlike EZH2 inhibition, restores SATB1 expression, selectively abrogating the growth of primary Sezary cells more effectively than Romidepsin. Therefore, SATB1 acts as a tumor suppressor in mature T cells upon NOTCH1 deregulation, and inhibition of methyltransferases that silence SATB1 could address an unmet need for patients with mycosis fungoides and Sezary syndrome.

Tertiary Lymphoid Structures (TLS) are commonly identified in human tumors with improved outcome, but how they are orchestrated remains elusive. Here we show that silencing of the master genomic organizer Satb1 results in enhanced antigen-specific T Follicular Helper (TFH) differentiation. Increased TFH thereby promoted antigen-specific intra-tumoral CD19+B220+ B cell responses and spontaneous TLS assembly upon ovarian tumor challenge. Mechanistically, Satb1 deficiency drives increased TFH formation through de-repression of ICOS and PD-1. Accordingly, TGF-β1-driven downregulation of Satb1 licenses activated human CD4+ T-cells for enhanced antigen-specific T Follicular Helper (TFH) differentiation. Furthermore, Satb1 deficiency abrogates the generation of PD-1highCXCR5+Foxp3+ T Follicular Regulatory (TFR) cells during the TFH differentiation process. Importantly, functional TFH cell accumulation, in the absence of Satb1 specifically in CD4+ T cells, resulted in corresponding isotype-switched B cell responses and spontaneous formation of TLS, while B cell depletion accelerated malignant progression. Our results indicate that the formation of TLS in cancer depends on enhanced B cell responses driven by TFH cells generated through Satb1 down-regulation.

The relationship between stem-like, tissue resident memory (TRM) and exhausted T cells at tumor beds is incompletely understood. We found that more than 50% of the CD8+ T cells in human ovarian carcinomas are TRM cells. RNA seq of TRM and their re-circulating counterparts infiltrating multiple human ovarian carcinomas showed a very distinctive phenotype, characterized by co-upregulation of effector and exhaustion markers, along with increased clonality and marked proliferative and lipid metabolism signatures. Interestingly, both populations showed very little overlap in TCR repertoire. Single cell analysis of TRM population showed that this is a heterogeneous population composed of different clusters defined by gene expression and TCR repertoire. A distinctive cluster shows higher expression of cytotoxic mediators and exhaustion markers, along with higher clonality and higher proliferation rate, with a TCR repertoire shared by CD103+TCF7+ stem-like cells. Single cell ATACseq showed that TRM and their counterpart have different chromatin structure and, within both populations also appear variables epigenetic landscapes. In addition, the presence of TRM is correlated with better prognosis, while tumor antigen-specific TRM T cells transferred into syngeneic tumor-bearing mice were more effective at delaying tumor growth than their tumor antigen-specific re-circulating counterparts. Together, our data indicate that productive immune pressure against malignant progression depends on a subset of CD8+ stem-like T cells differentiating into a narrow cluster of TRM T cells of ~300 TCR clones, which represent true tumor antigen-specific effector lymphocytes.

Due to their cytotoxic activities, many anticancer drugs cause extensive damage to the intestinal mucosa and have antibiotic activities. Here, we show that cisplatin induces significant changes in the repertoire of intestinal commensal bacteria that exacerbate mucosal damage. Restoration of the microbiota through fecal-pellet gavage drives healing of cisplatin-induced intestinal damage. Bacterial translocation to the blood stream is correspondingly abrogated, resulting in a significant reduction in systemic inflammation, as evidenced by decreased serum IL-6 and reduced mobilization of granulocytes. Mechanistically, reversal of dysbiosis in response to fecal gavage results in the production of protective mucins and mobilization of CD11b+ myeloid cells to the intestinal mucosa, which promotes angiogenesis. Administration of Ruminococcus gnavus, a bacterial strain selectively depleted by cisplatin treatment, could only partially restore the integrity of the intestinal mucosa and reduce systemic inflammation, without measurable increases in the accumulation of mucin proteins. Together, our results indicate that reconstitution of the full repertoire of intestinal bacteria altered by cisplatin treatment accelerates healing of the intestinal epithelium and ameliorates systemic inflammation. Therefore, fecal microbiota transplant could paradoxically prevent life-threatening bacteremia in cancer patients treated with chemotherapy.

Introduction: Many anti-tumor therapies induce apoptotic cell death in order to cause tumor regression. Paradoxically, apoptotic cells are also known to promote wound healing, cell proliferation, and tumor cell repopulation in multicellular organisms. We aimed to characterize the nature of the regenerative signals concentrated in the micro milieu of dead and dying cells. Methods: Cultures of viable melanoma B16F10 cells, mouse fibroblasts and primary human fibroblast-like synoviocytes(FLS) in the presence of dead and dying cells,its supernatants or purified agonists and antagonists were used to evaluate the stimulation of proliferation. Viable cell quantification was performed by either flow cytometry of harvested cells or by crystal violet staining of adherent cells. High-performance liquid chromatography (HPLC) and liquid chromatography coupled with mass spectrometry (LC-MS) of cell supernatants were deployed to identify the nature of growth promoting factors.Co-implantation of living cells in the presence of supernatants collected from dead and dying cells and specific agonists was used to evaluate tumor growth in vivo. Results: The stimulation of proliferation of few surviving cells by bystander dead cells was confirmed for melanomacells,mouse fibroblasts and primary FLS. We found that small soluble molecules present in the protein free fraction of supernatants of dead and dying cells were responsible for the promotion of proliferation. The nucleoside inosine released by dead and dying cells acting via adenosine receptors was identified as putative inducer of proliferation of surviving tumor cells after irradiation and heat treatment. Conclusion: Inosine released by dead and dying cells mediates tumor cell proliferation via purinergic receptors. Therapeutic strategies surmountingthis pathwaymay help to reduce the rate of recurrence after radio- and chemotherapy.

Tumours evade immune control by creating hostile microenvironments that perturb T cell metabolism and effector function 1?4 . However, it remains unclear how intra-tumoral T cells integrate and interpret metabolic stress signals. Here we report that ovarian cancer?an aggressive malignancy that is refractory to standard treatments and current immunotherapies 5?8 ?induces endoplasmic reticulum stress and activates the IRE1α?XBP1 arm of the unfolded protein response 9,10 in T cells to control their mitochondrial respiration and anti-tumour function. In T cells isolated from specimens collected from patients with ovarian cancer, upregulation of XBP1 was associated with decreased infiltration of T cells into tumours and with reduced IFNG mRNA expression. Malignant ascites fluid obtained from patients with ovarian cancer inhibited glucose uptake and caused N-linked protein glycosylation defects in T cells, which triggered IRE1α?XBP1 activation that suppressed mitochondrial activity and IFNγ production. Mechanistically, induction of XBP1 regulated the abundance of glutamine carriers and thus limited the influx of glutamine that is necessary to sustain mitochondrial respiration in T cells under glucose-deprived conditions. Restoring N-linked protein glycosylation, abrogating IRE1α?XBP1 activation or enforcing expression of glutamine transporters enhanced mitochondrial respiration in human T cells exposed to ovarian cancer ascites. XBP1-deficient T cells in the metastatic ovarian cancer milieu exhibited global transcriptional reprogramming and improved effector capacity. Accordingly, mice that bear ovarian cancer and lack XBP1 selectively in T cells demonstrate superior anti-tumour immunity, delayed malignant progression and increased overall survival. Controlling endoplasmic reticulum stress or targeting IRE1α?XBP1 signalling may help to restore the metabolic fitness and anti-tumour capacity of T cells in cancer hosts. Fil: Song, Minkyung. Weill Cornell Medicine; Estados Unidos Fil: Sandoval, Tito A.. Weill Cornell Medicine; Estados Unidos Fil: Chae, Chang-Suk. Weill Cornell Medicine; Estados Unidos Fil: Chopra, Sahil. Weill Cornell Medicine; Estados Unidos Fil: Tan, Chen. Weill Cornell Medicine; Estados Unidos Fil: Rutkowski, Melanie R.. University of Virginia; Estados Unidos Fil: Raundhal, Mahesh. Dana Farber Cancer Institute; Estados Unidos. Harvard Medical School; Estados Unidos Fil: Chaurio, Ricardo A.. H. Lee Moffitt Cancer Center & Research Institute; Estados Unidos Fil: Payne, Kyle K.. H. Lee Moffitt Cancer Center & Research Institute; Estados Unidos Fil: Konrad, Csaba. Weill Cornell Medicine; Estados Unidos Fil: Bettigole, Sarah E.. Quentis Therapeutics Inc.; Estados Unidos Fil: Shin, Hee Rae. Quentis Therapeutics Inc.; Estados Unidos Fil: Crowley, Michael J. P.. Weill Cornell Graduate School of Medical Sciences; Estados Unidos Fil: Cerliani, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina Fil: Kossenkov, Andrew V.. The Wistar Institute; Estados Unidos Fil: Motorykin, Ievgen. Weill Cornell Medicine,; Estados Unidos Fil: Zhang, Sheng. Weill Cornell Medicine,; Estados Unidos Fil: Manfredi, Giovanni. Weill Cornell Medicine,; Estados Unidos Fil: Zamarin, Dmitriy. Memorial Sloan Kettering Cancer Center; Estados Unidos Fil: Holcomb, Kevin. Weill Cornell Medicine,; Estados Unidos Fil: Rodriguez, Paulo C.. H. Lee Moffitt Cancer Center & Research Institute; Estados Unidos Fil: Rabinovich, Gabriel Adrián. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina Fil: Conejo Garcia, Jose R.. H. Lee Moffitt Cancer Center & Research Institute; Estados Unidos Fil: Glimcher, Laurie H.. Dana Farber Cancer Institute; Estados Unidos. Harvard Medical School; Estados Unidos Fil: Cubillos-Ruiz, Juan R.. Weill Graduate School Of Medical Sciences; Estados Unidos. Weill Graduate School Of Medical Sciences; Estados Unidos

Introduction Many antitumor therapies induce apoptotic cell death in order to cause tumor regression. Paradoxically, apoptotic cells are also known to promote wound healing, cell proliferation, and tumor cell repopulation in multicellular organisms. We aimed to characterize the nature of the regenerative signals concentrated in the micromilieu of dead and dying cells. Methods Cultures of viable melanoma B16F10 cells, mouse fibroblasts, and primary human fibroblast-like synoviocytes (FLS) in the presence of dead and dying cells, their supernatants (SNs), or purified agonists and antagonists were used to evaluate the stimulation of proliferation. Viable cell quantification was performed by either flow cytometry of harvested cells or by crystal violet staining of adherent cells. High-performance liquid chromatography and liquid chromatography coupled with mass spectrometry of cell SNs were deployed to identify the nature of growth-promoting factors. Coimplantation of living cells in the presence of SNs collected from dead and dying cells and specific agonists was used to evaluate tumor growth in vivo. Results The stimulation of proliferation of few surviving cells by bystander dead cells was confirmed for melanoma cells, mouse fibroblasts, and primary FLS. We found that small soluble molecules present in the protein-free fraction of SNs of dead and dying cells were responsible for the promotion of proliferation. The nucleoside inosine released by dead and dying cells acting via adenosine receptors was identified as putative inducer of proliferation of surviving tumor cells after irradiation and heat treatment. Conclusion Inosine released by dead and dying cells mediates tumor cell proliferation via purinergic receptors. Therapeutic strategies surmounting this pathway may help to reduce the rate of recurrence after radio- and chemotherapy.

T Follicular Helper cells (TFH) provide both co-stimulation and stimulatory cytokines to B cells to facilitate affinity maturation, class switch recombination, and plasma cell differentiation within the germinal center. However, is not clear how TFH differentiation is regulated. We found that deficiency of the chromatin organizer Satb1 results in increased TFH formation in CD4Cre+Satb1flx/flx mice through up-regulation of the canonical TFH markers ICOS and PD-1 and suppression of Foxp3+PD-1highCXCR5+ T follicular regulatory (TFR) cells as well. Accordingly, CD4Cre+Satb1flx/flx mice, or RAG1−/− mice transferred with Satb1-deficient CD4+ T cells showed a dramatic accumulation of CD4+CXCR5+PD-1high upon ovarian tumor challenge, compared to their Satb1+ counterparts, which was associated with reduced tumor growth. Importantly, intratumoral administration of Satb1-deficient CD4+ T cells re-directed to target ovarian cancer cells through chimeric receptors, but not their Satb1+ counterparts, induce the formation of Tertiary Lymphoid Structures in most tumors. Conclusion Satb1 controls three mechanisms relevant for TFH differentiation and, subsequently, antigen-specific humoral responses; namely, PD- 1 expression, ICOS de-repression and TFR formation. Our results suggest a novel role for Satb1 as a major regulator of TFH differentiation and TLS during tumor formation.

The response of the immune system against dying and dead cells strongly depends on the cell death phenotype. Beside other forms of cell death, two clearly distinct populations, early apoptotic and secondary necrotic cells, have been shown to induce anti-inflammation/tolerance and inflammation/immune priming, respectively. Cytofluorometry is a powerful technique to detect morphological and phenotypical changes occurring during cell death. Here, we describe a new technique using AnnexinA5, propidiumiodide, DiIC1(5) and Hoechst 33342 to sub-classify populations of apoptotic and/or necrotic cells. The method allows the fast and reliable identification of several different phases and pathways of cell death by analysing the following cell death associated changes in a single tube: cellular granularity and shrinkage, phosphatidylserine exposure, ion selectivity of the plasma membrane, mitochondrial membrane potential, and DNA content. The clear characterisation of cell death is of major importance for instance in immunization studies, in experimental therapeutic settings, and in the exploration of cell-death associated diseases. It also enables the analysis of immunological properties of distinct populations of dying cells and the pathways involved in this process.

The presence of a solid tumor is the result of a complex balance between rejection, tolerance and regeneration in which the interactions of tumor cells with cells of the host immune system contribute strongly to the final outcome. Here we report on a model where lethally UVB-irradiated cells cause accelerated growth of viable tumor cells in vitro and in allogeneic immune competent mice. UVB-irradiated tumor cells alone did not form tumors and failed to induce tolerance for a second challenge with the same allogeneic tumor. Our data show an important role for dying cells in promoting accelerated tumor cell growth of a small number of viable tumor cells in a large inoculum of UVB-irradiated tumor cells. This occurs when viable and dying/dead tumor cells are in close proximity, suggesting that mobile factors contribute to growth promotion. The anti-inflammatory and growth promoting properties of apoptotic cells are based on several independent effects. UVB-irradiated apoptotic cells directly release a growth promoting activity and clearance by macrophages of apoptotic cells is accompanied by the secretion of IL10, TGFß, and PGE2. Growth promotion is even observed with dying heterologous cells implying a conserved mechanism. Future experiments should focus on the effects of dying tumor cells generated in vivo on the outgrowth of surviving tumor cells which is prone to have implications for cancer therapy.

Killing tumor cells is a central goal of non-surgical cancer therapy and induction of apoptosis in the malignant cells is the major strategy used to achieve it. However, this may have serious drawbacks, since apoptotic cells reportedly induce immunological tumor tolerance and activate a tumor repopulation program. The debate on which type of cell death has the most beneficial therapeutic effects in cancer treatment is intense and controversial. Stringently regulated, doxycycline-inducible transgene expression systems trigger cell death in a defined manner, which might help us find answers to these problems. A conditional suicide switch established transiently in Jurkat T-cells was used to test a set of pro-apoptotic proteins for their potency to induce cell death. The activated forms of caspase-3 (revCasp-3) and Bid (tBid) were very effective and, therefore, analyzed after stable integration into human leukemia and murine melanoma cell lines. Expression of either protein resulted in more than 95% cell death, but with strongly different kinetics. 85% cell death was observed if tBid and revCasp-3 were expressed for 4-6 hours and 18-24 hours, respectively. The human cell lines expressing these suicide switches can serve as cancer models in xeno-transplanted mice, the corresponding murine cell lines allow syngeneic and allogeneic murine cancer models to be established.

The reaction of hydrogen sulfide (H 2 S) with peroxynitrite (a key mediator in numerous pathological states) was studied in vitro and in different cellular models. The results show that H 2 S can scavenge peroxynitrite with a corresponding second order rate constant of 3.3±0.4×10 3 M −1 ·s −1 at 23°C (8±2×10 3 M −1 ·s −1 at 37°C). Activation parameters for the reaction (Δ H ‡ , Δ S ‡ and Δ V ‡ ) revealed that the mechanism is rather associative than multi-step free-radical as expected for other thiols. This is in agreement with a primary formation of a new reaction product characterized by spectral and computational studies as HSNO 2 (thionitrate), predominantly present as sulfinyl nitrite, HS(O)NO. This is the first time a thionitrate has been shown to be generated under biologically relevant conditions. The potential of HS(O)NO to serve as a NO donor in a pH-dependent manner and its ability to release NO inside the cells has been demonstrated. Thus sulfide modulates the chemistry and biological effects of peroxynitrite by its scavenging and formation of a new chemical entity (HSNO 2 ) with the potential to release NO, suppressing the pro-apoptotic, oxidative and nitrative properties of peroxynitrite. Physiological concentrations of H 2 S abrogated peroxynitrite-induced cell damage as demonstrated by the: (i) inhibition of apoptosis and necrosis caused by peroxynitrite; (ii) prevention of protein nitration; and (iii) inhibition of PARP-1 [poly(ADP-ribose) polymerase 1] activation in cellular models, implying that a major part of the cytoprotective effects of hydrogen sulfide may be mediated by modulation of peroxynitrite chemistry, in particular under inflammatory conditions.

Despite the emergence of immunotherapy for the treatment of cancer, many of the fundamental mechanisms which characterize tumors that are amenable to immunotherapy and/or drive superior endogenous anti-tumor immune responses likely remain uncharacterized. We have identified a single-nucleotide polymorphism, rs13205210, in the gene encoding UHRF1BP1 (UBP). This polymorphism is associated with a dramatic survival benefit in ovarian cancer patients. The function of the protein encoded by this gene remains elusive, however we demonstrate UBP-ablated ovarian tumor cells display global modulation of methylated cytosine, suggesting it has a role as an epigenetic integrator. Interestingly, this polymorphism is also associated with systemic lupus erythematosus, an immune-driven pathology. Accordingly, we demonstrate that human ovarian tumors with polymorphic UBP display increased frequency of activated CD8+ T cells, as well as a type I IFN signature. In vivo, inducible autochthonous murine ovarian tumors driven by oncogenic Kras and ablation of p53, in which UBP was conditionally deleted, demonstrated a significantly enhanced overall survival with a concomitant type I IFN and CXCR3-chemokine signature, as well as an enhanced T cell infiltrate compared to controls. RNA-seq analyses of UBP-deficient ovarian tumors revealed an elevation of inflammatory cytokines and the activation of canonical inflammatory pathways. Furthermore, ectopic expression of polymorphic human UBP in ovarian tumor cells drove elevated NF-kB signaling under inflammatory conditions. Overall our work suggests that UBP functions as a regulator of inflammation, which is unleashed in the polymorphic variant leading to enhanced anti-tumor immunity.

Despite the importance of Programmed Cell Death-1 (PDCD1/PD-1) in inhibiting T-cell effector activity, the mechanisms regulating its expression in anti-tumor lymphocytes remain poorly defined. Here we show that the chromatin organizer Special AT-rich Sequence-Binding Protein-1 (Satb1) is required to restrain activation-induced PD-1 expression. We demonstrate that, mechanistically, Satb1 physically interacts with a nucleosome remodeling deacetylase (NuRD) complex, which it recruits to Pdcd1 regulatory regions. This molecular complex thus drives histone de-acetylation and results in PD-1 repression in T cells. Accordingly, T-cell-specific Satb1 deficiency results in a 40-fold increase in PD-1 expression. Intriguingly, tumor-derived Transforming growth factor (Tgf)-β decreases Satb1 expression in T cells through binding of Smad Family Member (Smad) proteins to the Satb1 promoter, while Smad also competes with Satb1/NuRD for binding to Pdcd1 enhancers, cooperatively unleashing PD-1 expression in a Satb1-dependent manner. Consequently, Satb1-deficient tumor-reactive T cells lose their effector activity more rapidly than wild-type T cells at PD-L1+ tumor beds, but these differences are abrogated by sustained PD-L1 blockade. Therefore, we demonstrate that Satb1 is an epigenetic controller of PD-1 expression, and that Tgf-β signaling contributes to T cell dysfunction within the tumor microenvironment by inhibiting Satb1-mediated repression of PD-1.

Large amounts of dead and dying cells are produced during cancer therapy and allograft rejection. Depending on the death pathway and stimuli involved, dying cells exhibit diverse features resulting in defined physiological consequences for the host. It is not fully understood how dying and dead cells modulate the immune response of the host. To address this problem, different death stimuli were studied in B16F10 melanoma cells by inducible transgene expression of the pro-apoptotic active forms of caspase-3 (revCasp-3), Bid (tBid), and the Mycobacterium tuberculosis-necrosis inducing toxin (CpnTCTD). The immune outcome elicited for each death stimulus was assessed by evaluating the allograft rejection of melanoma tumors implanted subcutaneously in BALB/c mice immunized with dying cells. Expression of all proteins efficiently killed cells in vitro (>90%) and displayed distinctive morphological and physiological features as assessed by multiparametric flow cytometry analysis. BALB/c mice immunized with allogeneic dying melanoma cells expressing revCasp-3 or CpnTCTD showed strong rejection of the allogeneic challenge. In contrast, mice immunized with cells dying either after expression of tBid or irradiation with UVB did not, suggesting an immunologically silent cell death. Surprisingly, immunogenic cell death induced by expression of revCasp-3 or CpnTCTD correlated with elevated intracellular ROS levels at the time-point of immunization. Conversely, early mitochondrial dysfunction induced by tBid expression or UVB irradiation accounted for the absence of intracellular ROS accumulation at the time point of immunization. Although ROS inhibition in vitro was not sufficient to abrogate the immunogenicity in our allo-immunization model, we suggest that the time-point of ROS generation and intracellular accumulation may be an important factor for its role as DAMP in the development of allogeneic responses.

Overwhelming apoptosis combined with a deficiency in clearing apoptotic cells is thought to be an important etiopathogenic event in the autoimmune disease systemic lupus erythematosus (SLE). Lazy macrophages, complement or DNase I deficiency as well as insufficient natural IgM might be important factors leading to such a clearance deficiency. A defective clearance of apoptotic cells leads to the activation and maturation of plasmacytoid and myeloid dendritic cells (DCs) by material derived from secondary necrotic cells carrying modified autoantigens. This results in the presentation of autoantigens to autoreactive T and B cells in an immunogenic manner, thereby leading to autoantibody production, chronic inflammation and severe tissue damage. Since DC activation and IFN-α production by plasmacytoid dendritic cells play a critical role in the course of SLE pathogenesis, therapeutic intervention to end this vicious cycle might be a promising approach for treating the disease.

The sequel to the landmark article “The Hallmarks of Cancer” adds two emerging hallmarks and two enabling characteristics to the six original hallmarks (1). One emerging hallmark is the property of cancer cells to escape the immune system. Clinically apparent tumors arise as winners in a complex, hard-fought duel between cancer cell survival and eradication by the immune system.

Healthy cells exhibit an asymmetric plasma membrane with phosphatidylserine (PS) located on the cytoplasmic leaflet of the plasma membrane bilayer. Annexin A5-FITC, a PS binding protein, is commonly used to evaluate apoptosis in flow cytometry. PS exposed by apoptotic cells serves as a major 'eat-me' signal for phagocytes. Although exposition of PS has been observed after alternative stimuli, no clearance of viable, PS exposing cells has been detected. Thus, besides PS exposure, membranes of viable and apoptotic cells might exhibit specific characteristics. Here, we show that Annexin A5 binds in a cooperative manner to different types of dead cells. Shrunken apoptotic cells thereby showed the highest Hill coefficient values. Contrarily, parafomaldehyde fixation of apoptotic cells completely abrogates the cooperativity effect seen with dead and dying cells. We tend to speculate that the cooperative binding of Annexin A5 to the membranes of apoptotic cells reflects higher fluidity of the exposed membranes facilitating PS clustering.

The phagocytosis of apo cells in patients with systemic lupus erythematosus (SLE) has been shown to be impaired. Recognition and engulfment of dying cells are mediated by molecules and receptors that are also involved in cell adhesion. Therefore, we analysed the adhesion capabilities of MoMa from patients with SLE. We employed a colorimetric assay to quantify the adhesion of MoMa to various substrates. In addition, we evaluated the morphological characteristics of the MΦ adhesion cultures by phase contrast microscopy. We have observed various morphologic characteristics depending on the adhesive substrate. The typical spindle shaped MoMa adhered to plastic and a bigger and rounder formed MoMa adhered to immunoglobulin (Ig) coated surfaces. Quantitatively, adhesion to plastic was significantly decreased in some SLE patients. Those patients showing a defective adhesion to plastic simultaneously showed an enhanced adhesion to Ig coated surfaces. These findings suggest that there is an alteration in the integ...

CRP is an important inflammatory marker, however, CRP levels are relatively low in patients with SLE. In addition patients with SLE often display low activities and serum levels for DNase I and complement, respectively. Here we show that DNase I treatment of nec PBMC increased their binding of CRP. Consequently, reduced DNase I activity in patients with SLE may contribute to the impaired opsonisation by CRP of dead cells, exacerbating the clearance defect in SLE of apo and nec cells.

Apoptosis and necrosis reflect the program of cell death employed by a dying cell and the final stage of death, respectively. Whereas apoptosis is defined as a physiological, highly organized cell death process, necrosis is commonly considered to be accidental and uncontrolled. Physiological and weak pathological death stimuli preferentially induce apoptosis, while harsh non-physiological insults often immediately instigate (primary) necrosis. If an apoptosing cell transits into a phase of plasma membrane disintegration, this stage of death is referred to as secondary or post-apoptotic necrosis.Here, we present several conditions that stimulate primary and/or secondary necrosis and show that necrosis displays considerably different time courses. For subclassification of necrotic phenotypes we employed a flow cytometric single-tube 4-color staining technique including annexin A5-FITC, propidium iodide, DiIC1(5), and Hoechst 33342.

Transmission-blocking (TB) vaccines are considered an important tool for malaria control and elimination. Among all the antigens characterized as TB vaccines against Plasmodium vivax, the ookinete surface proteins Pvs28 and Pvs25 are leading candidates. These proteins likely originated by a gene duplication event that took place before the radiation of the known Plasmodium species to primates. We report an evolutionary genetic analysis of a worldwide sample of pvs28 and pvs25 alleles. Our results show that both genes display low levels of genetic polymorphism when compared to the merozoite surface antigens AMA-1 and MSP-1; however, both ookinete antigens can be as polymorphic as other merozoite antigens such as MSP-8 and MSP-10. We found that parasite populations in Asia and the Americas are geographically differentiated with comparable levels of genetic diversity and specific amino acid replacements found only in the Americas. Furthermore, the observed variation was mainly accumulated in the EGF2- and EGF3-like domains for P. vivax in both proteins. This pattern was shared by other closely related non-human primate parasites such as Plasmodium cynomolgi, suggesting that it could be functionally important. In addition, examination with a suite of evolutionary genetic analyses indicated that the observed patterns are consistent with positive natural selection acting on Pvs28 and Pvs25 polymorphisms. The geographic pattern of genetic differentiation and the evidence for positive selection strongly suggest that the functional consequences of the observed polymorphism should be evaluated during development of TBVs that include Pvs25 and Pvs28., Author Summary Plasmodium vivax is the most prevalent human malarial parasite outside Africa. The fact that patients can relapse due to the parasite dormant liver stages, among other biologic and epidemiologic characteristics of vivax malaria, facilitates the persistence of the disease in many endemic areas. These challenges have fueled the search for new control tools, including transmission blocking (TB) vaccines targeting the parasite sexual stages. Here we study the genetic diversity of two major TB vaccine antigens, Pvs25 and Pvs28. We show that these genes are relatively conserved worldwide but still harbor diversity that is not evenly distributed across the genes. These patterns are shared by the same proteins in closely related parasite species suggesting their functional importance. We also identify strong geographic differentiation between the circulating variants found in Asia and the Americas. Finally, evolutionary genetic analyses indicate that the observed variation in both genes could be maintained by natural selection. Thus, these polymorphisms may confer an adaptive advantage to the parasite. These results indicate that the genetic variation found in these genes and their geographic distribution should be considered by vaccine developers.

Neutrophil extracellular traps (NETs) are fibers of extracellular DNA released from neutrophils due to overwhelming phagocytic stimuli. The function of NETs is to trap and kill microbes to avoid spreading of potential pathogens. NETs are formed after encounter with various gram-positive and -negative bacteria but also in response to mediators causing sterile inflammation like interleukin-8 (IL-8), tumor necrosis factor (TNF), and phorbol myristate acetate (PMA). Here we show the formation of NETs (NETting) in response to monosodium urate (MSU) crystals as further model for sterile inflammation. We identified monocytes, neutrophils, and eosinophils as MSU phagocytosing cells. Basophils did not take up the crystals, instead they upregulated their activation marker CD203c after contact with MSU. Nevertheless, MSU crystals induced extracellular trap formation also in basophils, like in eosinophils and neutrophils, which phagocytose the crystals. In contrast, monocytes do not form NETs despite uptake of the MSU crystals. In contrast to the canonical stimuli like bacteria and PMA, MSU-induced NETosis was not abrogated by plasma. Our data show that MSU crystals induce extracellular DNA trap formation in all three granulocytes lineages (NETs, EETs, and BETs) but not in monocytes, and DNA externalization does not necessitate the uptake of the crystals.

The phagocytosis of apo cells in patients with systemic lupus erythematosus (SLE) has been shown to be impaired. Recognition and engulfment of dying cells are mediated by molecules and receptors that are also involved in cell adhesion. Therefore, we analysed the adhesion capabilities of MoMa from patients with SLE. We employed a colorimetric assay to quantify the adhesion of MoMa to various substrates. In addition, we evaluated the morphological characteristics of the MPhi adhesion cultures by phase contrast microscopy. We have observed various morphologic characteristics depending on the adhesive substrate. The typical spindle shaped MoMa adhered to plastic and a bigger and rounder formed MoMa adhered to immunoglobulin (Ig) coated surfaces. Quantitatively, adhesion to plastic was significantly decreased in some SLE patients. Those patients showing a defective adhesion to plastic simultaneously showed an enhanced adhesion to Ig coated surfaces. These findings suggest that there is an alteration in the integrin and/or FcgammaR mediated stimulation of MoMa from SLE patients and this alteration may be associated with the impaired clearance of apo cells seen in this disease.