Role of B Cells in Graft-Versus-Leukemia and Graft-Versus-Host Disease

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. SCI-3-SCI-3
Author(s):  
Jerome Ritz
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Chronic Gvhd ◽  
Histocompatibility Antigens ◽  
Minor Histocompatibility Antigens ◽  
Graft Versus Host ◽  
Graft Versus Leukemia ◽  
Donor T Cells

Abstract The clinical outcomes of allogeneic hematopoietic stem cell transplantation (HSCT) have steadily improved in the last two decades, but this remains a potentially toxic treatment approach and further improvements are needed. Both the benefits and potential toxicities of allogeneic HSCT derive from the replacement of the recipient’s immune system with donor cells. Donor T cells clearly play a critical role as the primary mediators of both graft-versus-leukemia (GVL) and graft-versus-host-disease (GVHD) after transplant. In this setting, donor T cells targeting tumor-specific antigens provide specific GVL activity and donor T cells targeting broadly expressed minor histocompatibility antigens (allo-antigens) lead to GVHD. Donor T cells targeting minor histocompatibility antigens with restricted expression on both normal and malignant hematopoietic cells in the recipient contribute to GVL as well as to the elimination of recipient hematopoietic cells and the establishment of full donor hematopoiesis. Although donor B cells do not contribute to acute GVHD, considerable evidence now suggests that donor B cells also play an important role in chronic GVHD (cGVHD). In male patients with female donors, Y chromosome encoded (HY) proteins represent a clinically relevant set of widely expressed minor histocompatibility antigens (mHA) that are frequently recognized by both donor T cells and B cells. HY antibodies typically develop four to eight months after HSCT and the development of HY antibodies is significantly associated with the development of cGVHD. Antibodies to autosomal mHA and tumor-associated antigens have also been detected. Development of antibodies to mHA has also been associated with a lower risk of relapse suggesting a role for donor B cells in GVL. Murine models have clearly demonstrated that donor B cell reconstitution after allogeneic HSCT contributes to the development of cGVHD. In one of these models, depletion of germinal center B cells prevents the development of bronchiolitis obliterans and other pathologic features of cGVHD. The homeostatic cytokine B-cell activating factor (BAFF) plays an important role in the regulation of donor B cell reconstitution. BAFF promotes B-cell proliferation, differentiation, and survival; but persistent, high levels of BAFF also support the survival of auto and allo-reactive B cells. Patients with cGVHD typically have delayed B-cell reconstitution and low numbers of circulating B cells associated with high levels of BAFF. A high BAFF to B-cell ratio promotes survival of antigen-activated B cells and prevents or delays the development of B-cell tolerance after transplant. The important role of B cells in cGVHD has been confirmed by numerous clinical reports demonstrating the efficacy of B-cell directed therapy with rituximab in patients with established cGVHD. Overall response rates of 40 to 70 percent have been reported, and clinical responses have been associated with reduced titers of allo-reactive antibodies and restoration of normal B-cell homeostasis, with increased numbers of circulating B cells and lower levels of BAFF after recovery from treatment. The efficacy of rituximab in the treatment of established cGVHD has led to recent studies evaluating rituximab as a prophylactic therapy for cGVHD. The results of single institution trials suggest that this may be an effective approach and further randomized multi-center trials evaluating the role of rituximab for cGVHD prophylaxis are currently in development. The efficacy of rituximab has also led to the evaluation of other B cell directed therapies in murine models. In particular, selective inhibitors of B cell signaling pathways have been developed and appear to be effective in preventing cGVHD in these model systems. Further evaluation of these new agents in the treatment and prevention of cGVHD is in development. Disclosures: Off Label Use: Rituximab - Use in treatment of chronic GVHD..

scholarly journals The role of antigen-presenting cells in triggering graft-versus-host disease and graft-versus-leukemia

Blood ◽  
2007 ◽  
Vol 110 (1) ◽  
pp. 9-17 ◽  
Author(s):  
Ronjon Chakraverty ◽  
Megan Sykes
Keyword(s):  
Graft Versus Host Disease ◽  
Acute Gvhd ◽  
Tissue Injury ◽  
Chronic Gvhd ◽  
Host Disease ◽  
Graft Versus Host ◽  
Graft Versus Leukemia ◽  
Donor T Cells ◽  
Antigen Presenting

After allogeneic blood or bone marrow transplantation, donor T cells interact with a distorted antigen-presenting cell (APC) environment in which some, but not all, host APCs are replaced by APCs from the donor. Significantly, host APCs are required for the priming of acute graft-versus-host disease (GVHD). Donor APCs play a lesser role in the induction of acute GVHD despite their predicted capacity to cross-present host antigens. In contrast, donor APCs may play a role in perpetuating the tissue injury observed in chronic GVHD. Host APCs are also required for maximal graft-versus-leukemia responses. Recent studies have suggested potential strategies by which the continued presence of host APCs can be exploited to prime strong donor immunity to tumors without the induction of GVHD.

scholarly journals β2- Adrenergic Signaling Regulates Graft Versus Host Disease after Allogenic Transplantation While Preserving Graft Versus Leukemia Effect

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1915-1915 ◽  
Author(s):  
Hemn Mohammadpour ◽  
Joseph L. Sarow ◽  
George L. Chen ◽  
Cameron R. MacDonald ◽  
Umesh Sharma ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd4 T Cells ◽  
Adrenergic Receptor ◽  
Graft Versus Host ◽  
Graft Versus Leukemia ◽  
Donor T Cells ◽  
Β2 Adrenergic Receptor ◽  
Ifn Γ

β2 adrenergic receptor signaling is a key regulator of various immune cells, including T cells; however, its role in T cell function in the context of graft versus host disease (GvHD) is poorly understood. We previously showed that housing mice at thermoneutral temperature (TT; 30°C), which reduces systemic adrenergic stress, increased the incidence and severity of GvHD after allogeneic hematopoietic cell transplant (allo-HCT) compared to mice housed at standard temperature (ST; 22°C) which exerts a mild but chronic adrenergic stress (Leigh et al J Immunol 2015). The increased incidence and severity of GvHD in TT mice can be reversed by the administration of a β2-adrenergic receptor (β2-AR) agonist, suggesting an important role of epinephrine and norepinephrine in allo-HCT outcome (Leigh et al., J. Immunol 2015; Mohammadpour et al J Immunol 2018). We investigated the mechanisms and downstream events of β2-AR signaling in donor T cells after allo-HCT by using β2-AR knockout (β2-AR-/-) mice and commercially available β2-AR agonists. The main goal here was to explore whether signaling through β2-AR in donor T cells could control GvHD incidence and severity without minimizing the graft-versus leukemia (GvL) effect. We utilized both a major MHC-mismatch C57B6 (H-2kb) into BALB/c (H-2kd) model and a MHC-matched, multiple minor histocompatibility antigen (miHA) mismatched B6 (H-2kb) into C3H/SW (H-2kb) model. Recipient BALB/c and C3H/SW WT mice were lethally irradiated with 850 and 1100 cGy respectively and injected by tail vein with T cell depleted bone marrow (TCD-BM) alone (3 ×106) or TCD-BM and splenic T cells derived from allogeneic WT or β2-AR-/- B6 donors (0.7 × 106 T cells in B6 → BALB/c and 1.5 × 106 in B6 → C3H/SW). We found that donor T cells express β2-AR after allo-HCT and that β2-AR expression on WT T cells plays an important role in controlling GvHD, as evidenced by less severe weight loss, and increased survival compared to mice receiving β2-AR-/- donor T cells (Figure 1A). Histopathologic examination showed that β2-AR-/- T cells induced more damage in the small and large intestine. To explore further the mechanism(s) by which β2-AR signaling controls the severity of GvHD, we used NanoString analysis and discovered that β2-AR-/- T cells have the Th1 phenotype with an increase in Tbx21, Ifng, Irf8 and Emoes genes, while WT CD4+ T cells had higher levels of Th2 and Treg associated genes, including Foxp3, Ptgs5, Tgfb2, Il10, Il21 and Il22. We also observed a significant increase in the inflammatory cytokines IFN-γ and IL-17 in β2-AR-/- CD4+ T cells from the spleen and liver on days 7 and 14 after allo-HCT as compared to WT T cells (Figure 1B), while the expression of IL-10 was significantly higher in WT T cells compared to β2-AR-/- T cells (P< 0.01). We next sought to determine whether GvL may be affected by use of long acting β2-AR agonist (Bambuterol) to control GvHD. Bambuterol was administered daily at a dose of 1mg/kg from day 0. We observed that Bambuterol controlled the severity and mortality of GvHD after allo-HCT in both major and minor mismatch mouse models, as evidenced by reduced weight loss and an improved clinical score and survival rate in mice receiving Bambuterol compared to vehicle (P<0.001). We showed that treatment increased the expression of IL-10 and decreased the expression of IFN-γ and IL-17 in CD4+ T cells. Interestingly, we found that β2-AR agonist treatment significantly increased the generation of myeloid derived suppressor cells (MDSCs) from WT BM without any effect on β2-AR-/- BM both in vitro and in vivo, suggesting an important role of β2-AR signaling in the generation of MDSCs. To investigate the effect of Bambuterol on GvL, the A20 lymphoma cell line was injected 4 hours before allo-HCT. Using two different doses of T cells (0.5 × 106 and 0.2 × 106) in B6 → BALB/c model, we found that Bambuterol preserved GvL by inducing CD44+ CD62L- NKG2D+ effector cells and CD44+ CD62L+ central memory cells. Since β2-AR agonists can affect cardiac function, we measured heart rate (HR) and blood pressure (BP) using a tail-cuff. There was no difference in BP and HR at day 21 and 28 after allo-HCT between mice receiving Bambuterol compared to mice receiving vehicle. In conclusion, these data reveal how β-AR signaling can influence donor T cell differentiation and function in murine GvHD models without decreasing GvL effect pointing to the feasibility of manipulation of β2-AR signaling to ameliorate clinical GvHD. Disclosures No relevant conflicts of interest to declare.

Mutual Activation and Expansion of Donor CD4 + T Cells and B Cells in Transplants Play a Critical Role in the Initiation of Chronic Graft-Versus-Host Disease.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1342-1342
Author(s):  
James Sundblom Young ◽  
Dongchang Zhao ◽  
Tangsheng Yi ◽  
Hongjun Liu ◽  
Defu Zeng
Keyword(s):  
T Cells ◽  
Cell Differentiation ◽  
B Cells ◽  
B Cell ◽  
Graft Versus Host Disease ◽  
Cd4 T Cells ◽  
Chronic Gvhd ◽  
B Cell Differentiation ◽  
T And B Cells ◽  
Graft Versus Host

Abstract Abstract 1342 Poster Board I-364 Chronic graft versus host disease (GVHD) is an autoimmune-like disease, in which both donor CD4+ T and B cells play important roles in the pathogenesis. However, it is unclear how donor CD4+ T and B cells interact in the context of chronic GVHD. In our current studies, we found that, in a new chronic GVHD model of MHC-matched DBA/2 donor to BALB/c host, depletion of donor CD4+ T cells in transplants prevented donor B220+ B cell upregulation of co-stimulatory molecules (i.e. B7.1, B7.2, and MHC II), prevented donor B cell differentiation into syndecan+ IgG anti-dsDNA autoantibody-producing plasma cells, and prevented the induction of chronic GVHD. In addition, we found that donor CD4+ T cells were able to drive both marginal zone B (AA4.1−B220+CD1dhiCD23lo) and follicular B (AA4.1−B220+CD23hiCD1dlo) cells to become IgG autoantibody-producing cells. On the other hand, depletion of donor B220+ B cells in transplants prevented expansion of donor-type CD4+ T cells that proliferated in response to donor DC stimulation, prevented the skewing of TCR CDR3-length of the donor CD4+ T cells as revealed by TCR spectratyping, and prevented induction of chronic GVHD. These results indicate that donor CD4+ T and B cells mutually activate each other in the chronic GVHD recipients; alloreactive donor CD4+ T cells activate and drive donor B cell differentiation into IgG autoantibody producing cells, in turn, donor B cells mediate the expansion and TCR-spreading of autoreactive donor CD4+ T cells. Therefore, donor CD4+ T and B cells in transplants orchestrate the development of chronic GVHD. This work is supported by NIH R01 AI066008. Disclosures No relevant conflicts of interest to declare.

scholarly journals Distribution of minor histocompatibility antigens HA-1, HA-2 and HA-8 in the Croatian population

2020 ◽  
Vol 3 (1) ◽  
pp. 29-33
Author(s):  
Ana Posavec ◽  
Renata Zunec
Keyword(s):  
T Cells ◽  
Allogeneic Bone ◽  
Histocompatibility Antigens ◽  
Complex Molecules ◽  
Hematopoietic Stem ◽  
Minor Histocompatibility Antigens ◽  
Graft Versus Host ◽  
Histocompatibility Complex ◽  
Hla Dr

Minor histocompatibility antigens (mHAgs) are polymorphic, endogenously synthetized products recognized by alloreactive T cells in the context of major histocompatibility complex molecules. Recipients of allogeneic bone marrow grafts run the risk of graft-versus-host disease (GvHD), even when the donor is an HLA-identical sibling. This may be caused by disparities in mHAgs between the donor and the recipient, with the antigen present in the recipient and not in the donor. In such cases, T cells in the transplanted donor marrow respond to the recipient’s mHAgs. We determined the allele, genotype and phenotype frequencies for mHAgs HA-1, HA-2 and HA-8 in 102 healthy, unrelated individuals previously typed for HLA-A, HLA-B and HLA-DR. We compared the results with existing studies in other populations and found no significant differences between allele, genotype and phenotype frequencies in the Croatian population and frequencies reported for Caucasian population. The results presented will be used for further studies investigating the role of mHAgs in hematopoietic stem cell transplantation.

scholarly journals Prevention of Chronic Gvhd By Targeting Xbp-1 Genetically or Pharmacologically in Mice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4541-4541
Author(s):  
Steven D Schutt ◽  
Chih-Hang Anthony Tang ◽  
Yongxia Wu ◽  
David A Bastian ◽  
Juan Del Valle ◽  
...  
Keyword(s):  
T Cells ◽  
Stress Response ◽  
B Cells ◽  
Er Stress ◽  
B Cell ◽  
Chronic Gvhd ◽  
Critical Role ◽  
Conditional Knockout ◽  
Er Stress Response

Abstract Inhibition of the endoplasmic reticulum (ER) stress response via blockade of inositol-requiring enzyme-1α (IRE-1α) is currently a promising therapeutic strategy to treat B-cell leukemia, lymphoma, and myeloma. Because B cells play an important role in the development of chronic graft-versus-host disease (cGVHD), we hypothesize that the ER stress response contributes to B-cell function and pathogenicity in cGVHD. Here, we report that the ER stress response mediated by IRE-1α and its target X-box binding protein-1 (XBP-1) plays a critical role in cGVHD pathophysiology and represents a potential therapeutic target to prevent cGVHD. We tested the role of XBP-1 specifically in B cells by testing XBP-1 conditional knockout B cell grafts (XBP1fl/flCD19Cre+) in two mouse models of cGVHD. In the first model (B6 to BALB/c), recipients given XBP-1-deficient donor grafts showed significantly reduced cGVHD clinical scores, which were associated with reduced frequencies of donor-derived CD4 helper T cells within the lungs compared to the recipients of XBP-1fl/flCD19Cre- littermate donor grafts. XBP-1-deficient B cells produced significantly higher levels of IL-10 compared to WT control B cells after activation ex vivo. In the second model (B6 to B10.BR), the conversion of donor B cells to plasma cells (B220+CD38+CD138+) was reduced in both the spleens and lungs of recipients transplanted with XBP1fl/flCD19Cre+ grafts compared to those of the recipients given XBP1fl/flCD19Cre- grafts. Recipients given XBP1fl/flCD19Cre+ grafts also showed significantly higher total splenocytes and vastly increased splenic B-cell populations when compared with the recipients of XBP1fl/flCD19Cre- grafts. To expand on these findings, we tested if systemic XBP-1 blockade via a novel IRE-1α inhibitor, B-I09, would attenuate cGVHD. In a cutaneous model of cGVHD (B10.D2 to BALB/c), we found that prophylactic administration of B-I09 significantly reduced clinical features of cGVHD compared to vehicle controls (Fig. 1A). Validating these findings, hematoxylin and eosin stained skin sections of B-I09-treated mice had significantly lower pathology scores compared to vehicle controls (Fig. 1B). Isolated skin lymphocytes from recipients treated with B-I09 showed significant reductions in donor derived T cells and DCs compared to those treated with vehicle controls (Fig. 1C and D). Taken together, our findings reveal a novel role of the IRE-1α/XBP-1 pathway of the ER stress response in cGVHD pathophysiology and provide a readily translatable strategy to prevent the development of cGVHD in the clinic. Disclosures No relevant conflicts of interest to declare.

scholarly journals Aberrant B-cell homeostasis in chronic GVHD

Blood ◽  
2015 ◽  
Vol 125 (11) ◽  
pp. 1703-1707 ◽  
Author(s):  
Stefanie Sarantopoulos ◽  
Jerome Ritz
Keyword(s):  
B Cells ◽  
B Cell ◽  
Chronic Gvhd ◽  
Cell Receptor ◽  
Cell Homeostasis ◽  
Graft Versus Host ◽  
B Cell Homeostasis ◽  
Immune Pathology ◽  
New Strategies

Abstract Recent studies have compelled further interest in the potential pathological role of B cells in chronic graft-versus-host disease (cGVHD). In patients with cGVHD, B cells are activated and primed for survival via B-cell activating factor and B-cell receptor–associated pathways. Understanding the signaling pathways that drive immune pathology in cGVHD will facilitate the development of new strategies to selectively target aberrantly activated B cells and restore normal B-cell homeostasis after allogeneic stem cell transplantation.

scholarly journals Recent Metabolic Advances for Preventing and Treating Acute and Chronic Graft Versus Host Disease

2021 ◽  
Vol 12 ◽  
Author(s):  
Fathima A. Mohamed ◽  
Govindarajan Thangavelu ◽  
Stephanie Y. Rhee ◽  
Peter T. Sage ◽  
Roddy S. O’Connor ◽  
...  
Keyword(s):  
Fatty Acids ◽  
T Cells ◽  
B Cells ◽  
Immune Responses ◽  
Graft Versus Host Disease ◽  
Chronic Gvhd ◽  
Conditioning Regimen ◽  
Graft Versus Host ◽  
Helper Cells ◽  
Donor T Cells

The therapeutic efficacy of allogeneic hematopoietic stem cell transplantation (allo-HSCT) is limited by the development of graft-versus-host disease (GVHD). In GVHD, rigorous pre-conditioning regimen resets the immune landscape and inflammatory milieu causing immune dysregulation, characterized by an expansion of alloreactive cells and a reduction in immune regulatory cells. In acute GVHD (aGVHD), the release of damage- and pathogen- associated molecular patterns from damaged tissue caused by the conditioning regimen sets the stage for T cell priming, activation and expansion further exacerbating tissue injury and organ damage, particularly in the gastrointestinal tract. Studies have shown that donor T cells utilize multiple energetic and biosynthetic pathways to mediate GVHD that can be distinct from the pathways used by regulatory T cells for their suppressive function. In chronic GVHD (cGVHD), donor T cells may differentiate into IL-21 producing T follicular helper cells or tissue resident T helper cells that cooperate with germinal center B cells or memory B cells, respectively, to produce allo- and auto-reactive antibodies with subsequent tissue fibrosis. Alternatively, donor T cells can become IFN- γ/IL-17 cytokine expressing T cells that mediate sclerodermatous skin injury. Patients refractory to the first line standard regimens for GVHD treatment have a poor prognosis indicating an urgent need for new therapies to restore the balance between effector and regulatory immune cells while preserving the beneficial graft-versus-tumor effect. Emerging data points toward a role for metabolism in regulating these allo- and auto-immune responses. Here, we will discuss the preclinical and clinical data available on the distinct metabolic demands of acute and chronic GVHD and recent efforts in identifying therapeutic targets using metabolomics. Another dimension of this review will examine the changing microbiome after allo-HSCT and the role of microbial metabolites such as short chain fatty acids and long chain fatty acids on regulating immune responses. Lastly, we will examine the metabolic implications of coinhibitory pathway blockade and cellular therapies in allo-HSCT. In conclusion, greater understanding of metabolic pathways involved in immune cell dysregulation during allo-HSCT may pave the way to provide novel therapies to prevent and treat GVHD.

scholarly journals The allogeneic bisection of carrier-specific enhancement of monoclonal B-cell responses.

1975 ◽  
Vol 142 (5) ◽  
pp. 1165-1179 ◽  
Author(s):  
S K Pierce ◽  
N R Klinman
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Immunoglobulin Class ◽  
Cell Responses ◽  
Cell Clones ◽  
Ia Antigens ◽  
Collaborative Interactions ◽  
B Cell Responses

The ability of T cells to enhance the response of syngeneic and allogeneic B cells to thymus-dependent hapten-carrier conjugates was analyzed. This analysis was carried out on individual primary B cells in splenic fragment cultures derived from irradiated reconstituted mice. This system has several advantages: (a) the response of the B cells is entirely dependent on carrier priming of the irradiated recipient; (b) this B-cell response can be quantitated in terms of the number of responding cells; and (c) very small B-cell responses can be readily detected and analyzed. The results indicate that the majority of hapten-specific B cells were stimulated in allogeneic and syngeneic recipients only if these recipients were previously carrier primed. The number of B cells responding in carrier-primed allogeneic recipients was 60-70% of that in syngeneic carrier-primed recipients. The antibody-forming cell clones resulting from B cells stimulated in the allogeneic environment produced small amounts of antibody and antibody solely of the IgM immunoglobulin class, while the larger responses in syngeneic recipients were predominantly IgG1 or IgM plus IgG1. The capacity of collaborative interactions between carrier-primed T cells and primary B cells to yield IgG1 antibody-producing clones was shown to be dependent on syngeny between these cells in the H-2 gene complex. It is concluded that: (a) B cells can be triggered by T-dependent antigens to clone formation through collaboration with T cells which differ at the H-2 complex as long as these T cells recognize the antigen; (b) the immunoglobulin class produced by the progeny of stimulated B cells generally depends on the nature of the stimulatory event rather than the nature of the B cell itself; and (c) stimulation to IgG1 production is dependent on syngeny between the collaborating T and B cells probably within the Ir-1A region. The role of the Ia antigens in the formation of IgG1-producing clones is not yet clear; Ia identity could permit IgG1 production or, conversely, nonidentity of Ia could induce all allogeneic interactions which prohibit IgG1 production.

RhoF GTPase Transcription Is Upregulated in Germinal Center B-Cells, Shows Altered Expression in Malignant B-Cells, and Interacts with the ATM Pathway.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 285-285
Author(s):  
Launce G. Gouw ◽  
N. Scott Reading ◽  
David K. Crockett ◽  
Philippe Szankasi ◽  
Megan S. Lim ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
De Novo ◽  
Cell Lymphoma ◽  
Lymphocyte Subpopulations ◽  
Interacting Proteins ◽  
Tissue Samples

Abstract Follicular lymphoma (FL) is the most common low-grade B-cell non Hodgkin lymphoma in the Western hemisphere. A significant proportion of FL undergo histologic transformation to diffuse large B-cell lymphoma (DLBCL). Using cDNA microarray analysis, we identified an expressed sequence tag GI#10952525 consistently differentially expressed in transformed follicular lymphomas (tFL). This was characterized as RhoF, a novel member of the Rho family. Rho GTPases play central roles in cytoskeletal dynamics, cell-cell interactions, and intracellular signaling pathways involved in migration, proliferation and survival. Dysregulation of Rho proteins are key events implicated in tumorigenesis. To define the role of RhoF in lymphocyte physiology and lymphoma transformation, we assessed its expression across phenotypically defined lymphocyte subpopulations, using quantitative real-time PCR. We determined relative RhoF levels in immunomagnetic bead purified normal lymphoid subpopulations [naïve B-cells, memory B-cells, germinal center B-cells and T-cells], reactive lymphoid tissues (n=5), cell lines [derived from t(14;18) tFL (n =3), de novo DLBCL (n=7), and T-cell malignancy (n=3)] and tissue from primary human lymphoid neoplasms [FL (n=5), de novo DLBCL (n=5), tFL (n=5), CLL/SLL (n=4), anaplastic large cell lymphoma (n=8), mantle cell lymphoma (n=5), and T-cell acute lymphoblastic leukemia (n=5)]. RhoF was expressed at significantly higher levels in B-cells relative to T-cells. We saw this pattern in purified lymphocyte subpopulations, in cell lines, and in primary lymphoma tissue samples. Notably, we detected elevated levels of RhoF transcript in B-cells of germinal center (GC) origin, both in the reactive and neoplastic samples of GC-derived B-cells. The highest transcriptional levels of RhoF were in malignant B-cells of GC origin; both in heterogeneous primary tissue samples and in homogeneous tissue culture preparations. To investigate its functional role, we cloned RhoF into a vector coding for a C-terminal polyhistidine- and V5 epitope-tag. We expressed the constructs in HEK 293T cells, and purified the RhoF-containing complexes using a tandem affinity purification approach. We ran cell lysates through a nickel column; non-interacting proteins were washed off under native conditions and the bound RhoF complexes eluted with imidazole. Eluate was immunoprecipitated with sepharose-bound anti-V5 antibody. Immunoprecipitated complexes were denatured and resolved by 1D-PAGE. Unique bands representing RhoF interacting proteins were isolated and enzymatically cleaved with trypsin. Resultant peptides underwent liquid chromatography and tandem mass spectrometry. Data were searched against the NCBI nr.FASTA nonredundant protein database using the SEQUEST algorithm and false positive rates determined with INTERACT and ProteinProphet. Among several putative RhoF interactors, we identified ATM as an important RhoF binding partner. In conclusion, our demonstration of the differential expression of RhoF in GC-derived cells and its upregulation in tFL provide evidence for a connection between the role of this novel protein in B-cell development and malignancy. In addition, evidence of an association between RhoF and ATM may provide a link between DNA repair, cell cycle control and morphological dynamics.

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