scholarly journals Impaired B Lymphopoiesis Concurrent with Marrow Infiltration By Allogeneic Donor T-Cells: Further Evidence for Bone Marrow Graft-Versus-Host Disease

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3148-3148
Author(s):  
Armin Rashidi ◽  
Tandon Bevan ◽  
Amanda F. Cashen
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Count ◽  
Cell Infiltration ◽  
B Lymphopoiesis ◽  
Cell Recovery ◽  
Marrow Graft ◽  
T Cell Infiltration

Abstract Background: The recently introduced concept of bone marrow graft-versus-host disease (BM-GvHD) representing destruction of the host hematopoietic niche in the marrow by allogeneic donor T-cells is loosely defined. Otherwise unexplained B lymphocytopenia and other cytopenias of unclear etiology that frequently occur in the early post-transplant period are often attributed to BM-GvHD. B lymphocytopenia can co-exist with systemic GvHD, supporting the theory of concurrent donor T-cell-induced damage to the marrow causing suppressed B lymphopoiesis. However, demonstration of such correlations has been difficult in humans due to the lymphotoxic effect of steroids which are the frontline therapy for GvHD. The best evidence comes from a recent study showing a correlation between delayed recovery of B lymphopoiesis and donor T-cell infiltration in the marrow 3-4 weeks post-transplant. The potential confounding effect of steroids was not evaluated. The purpose of the present study was to assess whether there is significant donor T-cell infiltration in the marrow at the time of B lymphocytopenia in carefully selected, fully chimeric allo-SCT recipients on minimal or no steroids and with minimal or no systemic GvHD. Methods: A total of 11 patients who underwent allo-SCT for myeloid malignancies were retrospectively studied. Inclusion criteria were: (i) bone marrow biopsy available on days 90-100 or 170-190 post-SCT concurrent with peripheral blood B-cell count using flow cytometry, (ii) full donor chimerism at the time of bone marrow biopsy, (iii) no B lymphodepleting therapy post-SCT, (iv) not on more than 15 mg/d of prednisone on the day of measurement, (v) no GvHD other than acute stage I skin GvHD, and (vi) delayed B-cell recovery defined as <10 CD19+ B-cells/µl on days 90-100 or <100 cells/µl on days 180-200. Peripheral B-cell count measurements were not due to specific clinical indications and were either based on the treating physician's routine practice or the protocols patients were enrolled to. Similar to previous studies, increased T-cell infiltration was defined as ≥5% of total nucleated cells in the core determined by anti-CD3 antibody labeling in a fully chimeric recipient. We determined the frequency of increased marrow T-cell infiltration (as a marker of acute BM-GvHD) on the same day when delayed B-cell recovery (as a marker of impaired B lymphopoiesis) was diagnosed. Results: 11 patients (10 males) with a median (range) age of 60 (32-67) years were studied. Measurements were made between days 90-100 and 170-190 post-SCT in 4 and 7 patients, respectively. The underlying diagnosis was acute myeloid leukemia (n = 7) or myelodysplastic syndrome (n = 4). The donor was a matched sibling (n = 1), matched unrelated donor (n = 4), or haploidentical donor (n = 6). Conditioning was ablative in 5 patients. Leukopenia, anemia, and thrombocytopenia were present in 3, 9, and 11 patients, respectively. The median (range) B-cell count on days 90-100 and 180-200 was 7 (0-9) and 19 (0-65) cells/µl, respectively. All patients had ≥5% T-cells/µl in the concurrent core biopsy with one exception. This patient had zero B-cells on day 180 but no evidence of concurrent BM-GvHD while on 12 mg/d of prednisone for appetite stimulation. Conclusions: Using a carefully selected cohort of fully chimeric allo-SCT recipients with delayed B lymphopoiesis, on no or minimal amounts of steroids, and with minimal or no systemic GvHD, we demonstrated a high frequency of concurrent increased marrow T-cell infiltration. These results support the recently introduced concept of BM-GvHD and highlight its negative effect on B lymphopoiesis. We show that bone marrow damage by allogeneic T-cells can occur even in the absence of systemic GvHD. Given the difficulties in quantification of marrow T-cells on the core, a more reproducible definition for BM-GvHD is needed. Disclosures No relevant conflicts of interest to declare.

Acute Bone Marrow GvHD Is Associated With Delayed B Cell Neogenesis and Impaired Natural Antibody Response After Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 4605-4605
Author(s):  
Angela Mensen ◽  
Korinna Jöhrens ◽  
Ioannis Anagnostopoulos ◽  
Sonya Demski ◽  
Christoph Ochs ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Infiltration ◽  
Strong Increase ◽  
Cell Recovery ◽  
T Cell Infiltration ◽  
Transitional B Cells

Graft-versus-host disease (GvHD) and severe infections are main complications limiting the success of allogeneic hematopoietic stem cell transplantion (alloHSCT). Delayed B cell reconstitution followed by B cell immune dysfunction considerably contributes to an increased risk for life-threatening infections. Several studies have shown that B cell regeneration is impaired in patients with systemic GvHD. Bone marrow (BM) suppression is often observed in parallel as GvHD symptoms appear suggesting the BM as a target of GvHD. Thus far, little is known about mechanisms of BM dysfunction during GvHD in alloHSCT patients. In this study, we investigated the reconstitution kinetics of peripheral blood B cell subsets in adult acute leukemic patients (n=52) before and within six months after alloHSCT by flow cytometry and correlated the data with RT-PCR quantified numbers of kappa-deleting-recombination-excision-circles (KREC), which are stable episomal plasmids generated during BM B cell development. Furthermore, we determined specific B cell antibody responses after in vitrostimulation with CpG, CD40L and T cell cytokines by EliSPOT analysis. To investigate BM as a direct target of allo-reactive T cells we performed histopathological stainings of BM biopsy samples obtained 3-4 weeks after alloHSCT. T cells were detected by specific anti-CD3 antibody staining and osteoblasts were morphologically evaluated. We observed in all patients a profound B cell immune deficiency already pre-transplant that proceeded within the first months post alloHSCT (mean B cells/ml blood±SEM: 11±3 pretransplant, 3±1 day14, 3±1 day28 post alloHSCT; 83±13 healthy control). Onset of B cell reconstitution is characterized by transitional B cell recovery representing the first B cell subset which emigrates from the BM. B cell reconstitution occurred either early (37% of patients) with a strong increase of transitional B cells between days 60-90 (mean transitional B cells/ml blood±SEM: Day 60, 36±10) or late (33% of patients) with delayed recovering transitional B cells (Day 180, 5±2). KREC copy numbers correlated highly positive and significantly with transitional B cell numbers (Spearman 0.94, p=0.017). Less correlation was obtained with naïve and CD27+ memory B cell recovery. Delayed onset of B cell reconstitution was significantly associated with both presence of systemic acute GvHD and full-intensity conditioning therapy (GvHD 71% vs non-GvHD 32%, Fisher´s exact p=0.044; full-intensity 41% vs reduced-intensity 5%, p=0.016). Supporting the hypothesis of bone marrow GvHD we could show a stronger infiltration of CD3+ T cells in the BM in late than in early recovering patients (≥5% T cell infiltration: 64% vs 17%, p=0.010). This increased T-cell infiltration was associated with reduced numbers of osteoblasts, known in mice to support B cell lymphopoiesis (no/few osteoblasts: 65% vs 17%, p=0.011). Impaired B cell lymphopoiesis further resulted in a delayed naïve and IgM memory B cell recovery compared to early recovering patients. No recovery of switched-memory B cells was seen for both patient groups within the analyzed time-period. Functionally, ex vivoactivation of patient B cells revealed higher numbers of IgM producing B cells specific for pneumococcal polysaccharide (PnP) at day 180 post alloHSCT in early than in late recovering patients. Polyclonal IgG producing B cells were significantly diminished in all patients. We conclude from these data, that early onset of B cell reconstitution is characterized by strong increase in regenerating transitional B cells within three months after alloHSCT. Herein, KREC appears as a suitable biomarker to monitor BM B cell output post-transplant. B cell regeneration is significantly delayed in patients showing increased occurrence of systemic acute GvHD and stronger T cell infiltration with loss of osteoblasts in the BM. Thus, delayed onset of B cell reconstitution might result from acute BM GvHD in which alloreactive T cells lead to an osteoblast niche destruction. Increased PnP specific IgM antibody responses are most likely result of higher numbers of early reconstituted transitional and IgM memory B cells but not naïve B cells that were shown not to produce IgM upon CpG stimulation (Capolunghi F et al. 2008). Thus, early B cell reconstitution might provide a first natural antibody immunity after alloHSCT, emphasizing the importance of a functional bone marrow niche. Disclosures: No relevant conflicts of interest to declare.

Bone marrow T-cell infiltration during acute GVHD is associated with delayed B-cell recovery and function after HSCT

Blood ◽  
2014 ◽  
Vol 124 (6) ◽  
pp. 963-972 ◽  
Author(s):  
Angela Mensen ◽  
Korinna Jöhrens ◽  
Ioannis Anagnostopoulos ◽  
Sonya Demski ◽  
Maike Oey ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
B Cell ◽  
Cell Infiltration ◽  
Cell Recovery ◽  
T Cell Infiltration ◽  
Cell Immunity ◽  
Key Points ◽  
Excision Circles ◽  
And Function

Key Points Donor T-cell infiltration of the bone marrow is associated with impaired B-cell immunity after allogeneic HSCT. Quantification of κ-deleting recombination excision circles as a biomarker for bone marrow B-cell output in different clinical episodes.

MyD88 in Donor Bone Marrow Cells Is Critical for Protection from Acute Intestinal Graft-Versus-Host Disease

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1094-1094
Author(s):  
Ji Young Lim ◽  
Sung-Eun Lee ◽  
Yoo-Jin Kim ◽  
Gyeongsin Park ◽  
Eun Young Choi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Graft Versus Host Disease ◽  
Cell Infiltration ◽  
Host Disease ◽  
Graft Versus Host ◽  
T Cell Infiltration ◽  
Donor Bone Marrow ◽  
Intestinal Gvhd

Abstract Allogeneic hematopoietic stem cell transplantation is an important therapeutic modality used to treat malignancies of hematopoietic origin, such as leukemia and lymphoma. However, development of graft-versus-host disease (GVHD) causes non-relapse mortality and substantial morbidity of recipients. Myeloid differentiation factor 88 (MyD88), a major adaptor mediating TLR signaling, is also known to deliver pro-inflammatory signals. Activation of inflammatory signaling through MyD88 plays a key role in the expansion of myeloid-derived suppressor cells (MDSC) which are a heterogeneous population of immature myeloid ells with anti-inflammatory activity. To explore the contribution of MyD88 expressed by donor bone marrow (BM) cells to development of GVHD, we induced GVHD using T-cell-depleted BM (TCD-BM) isolated from MyD88-deficient (MyD88KO) mice and T cells isolated from wild-type (WT) mice. We employed C57BL/6 (H-2b) → B6D2F1 (H-2b/d) mouse model of GVHD, which differ at major and minor histocompatibility loci. Lethally irradiated B6D2F1 recipient mice were transplanted with either T cell-depleted bone marrow (TCD BM, 5 x 106) from either WT or MyD88KO mice together with WT spleen T cells (1 x 106). Transplantation with MyD88KO TCD BM aggravated GVHD; serious gut damage was evident, with infiltration of T cells specifically into the intestines of recipients. GVHD hosts transplanted with MyD88KO TCD BM exhibited markedly reduced expansion of MDSC. GVHD aggravation after transplantation with MyD88KO TCD-BM, associated with high-level T cell infiltration into the intestine and insignificant expansion of MDSC, was reproduced in another minor histocompatibility mismatch model (C57BL/6 → BALB.B). We next examined allogeneic T cells in the spleens of GVHD hosts in terms of the expression levels of CCR9, which are known to be associated with T cell migration to the intestinal mucosa and the proportion of CCR9 positive cells in CFSE low CD8+ T cells was higher in recipients of MyD88KO TCD BM than WT controls. In parallel, the levels of CCL25 were more highly expressed in the gut of MyD88KO recipients than WT controls. Mixed leukocyte cultures of CFSE- labeled C57BL/6 T cells and irradiated B6DF1 feeder cells were prepared in the presence of MDSC isolated from MyD88KO or WT mice. T cells, co-incubated with MDSC isolated from MyD88KO BM, exhibited a greater extent of CFSE dilution and less Annexin V staining, compared to T cells co-incubated with cells from WT BM. Moreover, MDSC from recipients of MyD88KO TCD BM exhibited a reduced suppressive function, compared to their WT counterparts. Next, we determined whether insufficient expansion of and ineffective suppression by MDSC caused severe GVHD in recipients of MyD88KO TCD BM. Supplementation of transplanted mice with MDSC from WT mice, not from MyD88KO mice, attenuated the severity of GVHD and reduced intestinal T cell infiltration in recipients of MyD88KO TCD BM. To verify the importance of MyD88-mediated signaling by MDSC in protection against severe GVHD, we determined if transplantation with TCD-BM cells containing high levels of MDSC attenuated the severity of GVHD. Pre-treatment of BM donors with lipopolysaccharide increased the frequencies of MDSC and the amounts of MyD88 transcripts in TCD-BM transplant, and alleviated the severity of GVHD and intestinal T-cell infiltration. To explore whether MDSC expansion levels could be used to predict the severity of intestinal GVHD, the T/MDSC ratios were calculated in blood of patients at the time of engraftment and were significantly higher in patients with intestinal GVHD ≥ grade 2. In conclusion, we have shown that MyD88-dependent MDSC expansion from donor BM is critical for protection against fatal acute intestinal GVHD. Disclosures No relevant conflicts of interest to declare.

scholarly journals 49 In situ phenotypic analysis of T cells in the tumor microenvironment of a pre-clinical model of non-small cell lung cancer (NSCLC) by tissue sectioning and whole-mount immunofluorescence imaging

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A52-A52
Author(s):  
Elen Torres ◽  
Stefani Spranger
Keyword(s):  
T Cells ◽  
Spatial Distribution ◽  
T Cell ◽  
Spatial Location ◽  
Cell Infiltration ◽  
Cell Populations ◽  
Tumor Bearing ◽  
T Cell Infiltration ◽  
Volume Imaging ◽  
Whole Mount

BackgroundUnderstanding the interactions between tumor and immune cells is critical for improving current immunotherapies. Pre-clinical and clinical evidence has shown that failed T cell infiltration into lung cancer lesions might be associated with low responsiveness towards checkpoint blockade.1 For this reason, it is necessary to characterize not only the phenotype of T cells in tumor-bearing lungs but also their spatial location in the tumor microenvironment (TME). Multiplex immunofluorescence staining allows the simultaneous use of several cell markers to study the state and the spatial location of cell populations in the tissue of interest. Although this technique is usually applied to thin tissue sections (5 to 12 µm), the analysis of large tissue volumes may provide a better understanding of the spatial distribution of cells in relation to the TME. Here, we analyzed the number and spatial distribution of cytotoxic T cells and other immune cells in the TME of tumor-bearing lungs, using both 12 µm sections and whole-mount preparations imaged by confocal microscopy.MethodsLung tumors were induced in C57BL/6 mice by tail vein injection of a cancer cell line derived from KrasG12D/+ and Tp53-/- mice. Lung tissue with a diverse degree of T cell infiltration was collected after 21 days post tumor induction. Tissue was fixed in 4% PFA, followed by snap-frozen for sectioning. Whole-mount preparations were processed according to Weizhe Li et al. (2019) 2 for tissue clearing and multiplex volume imaging. T cells were labeled with CD8 and FOXP3 antibodies to identify cytotoxic or regulatory T cells, respectively. Tumor cells were labeled with a pan-Keratin antibody. Images were acquired using a Leica SP8 confocal microscope. FIJI3 and IMARIS were used for image processing.ResultsWe identified both cytotoxic and regulatory T cell populations in the TME using thin sections and whole-mount. However, using whole-mount after tissue clearing allowed us to better evaluate the spatial distribution of the T cell populations in relation to the tumor structure. Furthermore, tissue clearance facilitates the imaging of larger volumes using multiplex immunofluorescence.ConclusionsAnalysis of large lung tissue volumes provides a better understanding of the location of immune cell populations in relation to the TME and allows to study heterogeneous immune infiltration on a per-lesion base. This valuable information will improve the characterization of the TME and the definition of cancer-immune phenotypes in NSCLC.ReferencesTeng MW, et al., Classifying cancers based on T-cell infiltration and PD-L1. Cancer Res 2015;75(11): p. 2139–45.Li W, Germain RN, and Gerner MY. High-dimensional cell-level analysis of tissues with Ce3D multiplex volume imaging. Nat Protoc 2019;14(6): p. 1708–1733.Schindelin J, et al, Fiji: an open-source platform for biological-image analysis. Nat Methods 2012;9(7): p. 676–82.

scholarly journals Primary vulvar squamous cell carcinomas with high T cell infiltration and active immune signaling are potential candidates for neoadjuvant PD-1/PD-L1 immunotherapy

2021 ◽  
Vol 9 (10) ◽  
pp. e003671
Author(s):  
Kim E Kortekaas ◽  
Saskia J Santegoets ◽  
Liselotte Tas ◽  
Ilina Ehsan ◽  
Pornpimol Charoentong ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Signaling Pathways ◽  
T Cell Activation ◽  
Squamous Cell ◽  
Molecular Subtype ◽  
Cell Infiltration ◽  
Immune Signaling ◽  
T Cell Infiltration ◽  
Expression Of Genes

BackgroundA profound insight into the immune landscape of vulvar squamous cell carcinoma (VSCC) is lacking. Here, an in-depth interrogation of T cell infiltration, local immune contexture, signaling pathways and checkpoint molecule expression was performed in early-stage and late-stage VSCC.MethodsThe type, location, and infiltration pattern of T cells were studied in 109 patients with primary VSCC FIGO stage I–III. RNA expression of genes involved in immune oncology and oncogenic signaling pathways was analyzed in 40 VSCC, matched for prognostic clinicopathological variables, analyzed for HPV and p53 status, and selected based on T cell infiltration.ResultsHigh intraepithelial infiltration with CD4 or CD8 T cells was associated with longer overall and recurrence-free survival and formed an independent prognostic factor, outperforming molecular subtype and stage of the disease. Strong T cell infiltrated VSCC displayed a coordinated immune response reflected by a positive association between T cells and different lymphocyte and myeloid cell subsets. The expression of genes involved in the migration of T cells and myeloid cells, T cell activation and costimulation, interferon (IFN)-γ signaling, cytotoxicity and apoptosis was higher than in low infiltrated tumors. An active immune signaling profile was observed in all inflamed, part of the altered-excluded and not in altered-immunosuppressed or deserted VSCC. While several checkpoint molecules were overexpressed, only PD-L1 expression displayed discriminatory ability and clinical usefulness. High PD-L1 expression was detected in all inflamed and ~60% of the altered-excluded VSCC.ConclusionAn active immune signaling profile is present in 35% of primary FIGO I–III VSCCs, suggesting potential responsiveness to neoadjuvant PD-1/PD-L1 immunotherapy.

scholarly journals Spatiotemporal single-cell profiling of gastrointestinal GVHD reveals invasive and resident memory T cell states

2020 ◽  
Author(s):  
Victor Tkachev ◽  
James Kaminski ◽  
E. Lake Potter ◽  
Scott N. Furlan ◽  
Alison Yu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
Cd8 T Cell ◽  
Transcriptomic Analysis ◽  
Cell Infiltration ◽  
Cell Transplant ◽  
Gi Tract ◽  
Tissue Invasion ◽  
T Cell Infiltration

ABSTRACTOne of the central challenges in the field of allo-immunity is deciphering the mechanisms driving T cells to infiltrate and subsequently occupy target organs to cause disease. The act of CD8-dominated T cell infiltration is critical to acute graft-versus-host disease (aGVHD), wherein donor T cells become activated, tissue-infiltrating and highly cytotoxic, causing wide-spread tissue damage after allogeneic hematopoietic stem cell transplant (allo-HCT). However, in human and non-human primate studies, deconvolving the transcriptional programs of newly recruited relative to resident memory T cells in the gastrointestinal (GI) tract has remained a challenge. In this study, we combined the novel technique of Serial Intravascular Staining (SIVS) with single-cell RNA-Seq (scRNA-seq) to enable detailed dissection of the tightly connected processes by which T cells first infiltrate tissues and then establish a pathogenic tissue residency program after allo-HCT in non-human primates. Our results have enabled the creation of a spatiotemporal map of the transcriptional drivers of CD8 T cell infiltration into the primary aGVHD target-organ, the GI tract. We identify the large and small intestines as the only two sites demonstrating allo-specific, rather than lymphdepletion-driven T cell infiltration. The donor CD8 T cells that infiltrate the GI tract demonstrate a highly activated, cytotoxic phenotype while simultaneously rapidly developing canonical tissue-resident memory (TRM) protein expression and transcriptional signatures, driven by IL-15/IL-21 signaling. Moreover, by combining SIVS and transcriptomic analysis, we have been able to work backwards from this pathogenic TRM programing, and, for the first time, identify a cluster of genes directly associated with tissue invasiveness, prominently including specific chemokines and adhesion molecules and their receptors, as well as a central cytoskeletal transcriptional node. The clinical relevance of this new tissue invasion signature was validated by its ability to discriminate the CD8 T cell transcriptome of patients with GI aGVHD. These results provide new insights into the mechanisms controlling tissue infiltration and pathogenic CD8 TRM transcriptional programing, uncovering critical transitions in allo-immune tissue invasion and destruction.One sentence summaryFlow cytometric and transcriptomic analysis reveals coordinated tissue-infiltration and tissue-residency programs driving gastrointestinal aGVHD.

Adoptive T-Cell Therapy for B-Cell Acute Lymphoblastic Leukemia: Preclinical Studies

Blood ◽  
1999 ◽  
Vol 94 (10) ◽  
pp. 3531-3540 ◽  
Author(s):  
Angelo A. Cardoso ◽  
J. Pedro Veiga ◽  
Paolo Ghia ◽  
Hernani M. Afonso ◽  
W. Nicholas Haining ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Adoptive T Cell Therapy ◽  
Leukemic Cells ◽  
Acute Leukemias ◽  
T Cell Lines

We have previously shown that leukemia-specific cytotoxic T cells (CTL) can be generated from the bone marrow of most patients with B-cell precursor acute leukemias. If these antileukemia CTL are to be used for adoptive immunotherapy, they must have the capability to circulate, migrate through endothelium, home to the bone marrow, and, most importantly, lyse the leukemic cells in a leukemia-permissive bone marrow microenvironment. We demonstrate here that such antileukemia T-cell lines are overwhelmingly CD8+ and exhibit an activated phenotype. Using a transendothelial chemotaxis assay with human endothelial cells, we observed that these T cells can be recruited and transmigrate through vascular and bone marrow endothelium and that these transmigrated cells preserve their capacity to lyse leukemic cells. Additionally, these antileukemia T-cell lines are capable of adhering to autologous stromal cell layers. Finally, autologous antileukemia CTL specifically lyse leukemic cells even in the presence of autologous marrow stroma. Importantly, these antileukemia T-cell lines do not lyse autologous stromal cells. Thus, the capacity to generate anti–leukemia-specific T-cell lines coupled with the present findings that such cells can migrate, adhere, and function in the presence of the marrow microenvironment enable the development of clinical studies of adoptive transfer of antileukemia CTL for the treatment of ALL.

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