scholarly journals A natural model of immunologic tolerance. Tolerance to murine C5 is mediated by T cells, and antigen is required to maintain unresponsiveness.

1982 ◽  
Vol 156 (2) ◽  
pp. 567-584 ◽  
Author(s):  
D E Harris ◽  
L Cairns ◽  
F S Rosen ◽  
Y Borel
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Lymphoid Cells ◽  
Immunologic Tolerance ◽  
Secondary Response ◽  
Spleen Cells ◽  
Physiological Concentration ◽  
Significant Drop ◽  
Natural Form

A unique experimental model is described, where natural immunologic tolerance to a well-defined soluble native antigen (murine C5) is examined in congenic strains of mice that differ only by the presence or the absence of C5. A highly sensitive hemolytic assay was developed to detect nanogram amounts of C5 as well as an assay of anti-C5 inhibition of C5 hemolytic activity. The latter was more sensitive than immunodiffusion. Two reciprocal approaches were used to study the cellular basis of tolerance in irradiated hosts of either strain. In the first, lymphoid cells from either strain were transferred to irradiated B10.D2OSN hosts that were lacking C5 and so would not hinder detection of anti-C5 antibody upon challenge with murine C5. Second, lymphoid cells from either strain were transferred to irradiated B10.D2NSN hosts, whose native C5 provided the antigenic stimulus. The immune response of whole nonadherent spleen cell suspension as well as mixtures of T and B cells (separated on the basis of surface immunoglobulin) from either strain were studied. In addition, the duration of tolerance and the antigen requirement to maintain it in irradiated C5-deficient hosts repopulated with C5-sufficient spleen cells was examined. The positive control of irradiated C5-deficient hosts repopulated with syngeneic spleen cells showed a primary and secondary response to immunization. In contrast, C5-sufficient spleen cells failed to respond both in the primary and the secondary response. Because the unresponsiveness was not caused by antigen carryover and was not antigen specific, it represents central tolerance. In C5-sufficient irradiated hosts (where immunization was not required and antigen was present in natural form and physiological concentration), transfer of C5-deficient cells mediated a drop in C5 levels to 10-20% of that noted in unreconstituted controls. T and B cell mixing experiments from the two strains into deficient or sufficient hosts demonstrated that tolerance is T cell dependent and that C5-sufficient or -deficient B cells could cooperate with nontolerant C5-sufficient T cells to produce significant anti-C5 antibody or mediate a significant drop in C5 levels. In addition, the presence of antigen was necessary to maintain tolerance. In conclusion, these results show that (a) natural tolerance to C5 is an active process that is T cell dependent and requires the presence of antigen; (b) in this natural model, clonal abortion does not seem to occur; and (c) both tolerant and nontolerant B cells retain the capacity to produce autoantibody.

scholarly journals The role of H-2-linked genes in helper T cell function. VII. Expression of I region and immune response genes by B cells in bystander help assays.

1980 ◽  
Vol 152 (5) ◽  
pp. 1274-1288 ◽  
Author(s):  
P Marrack ◽  
J W Kappler
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Function ◽  
High Responder ◽  
Helper T Cells ◽  
Spleen Cells ◽  
Region Gene ◽  
Immunoglobulin Receptor

The mode of action by bystander helper T cells was investigated by priming (responder X nonresponder) (B6A)F1 T cells with poly-L-(Tyr, Glu)-poly-D,L-Ala--poly-L-Lys [(TG)-A--L] and titrating the ability of these cells to stimulate an anti-sheep red blood cell (SRBC) response of parental B cells and macrophages in the presence of (TG)-A--L. Under limiting T cell conditions, and in the presence of (TG)-A--L, (TG)-A--L-responsive T cells were able to drive anti-SRBC responses of high-responder C57BL/10.SgSn (B10) B cells and macrophages (M0), but not of low-responder (B10.A) B cells and M0. Surprisingly, the (TG)-A--L-driven anti-SRBC response of B10.A B cells was not restored by addition of high-responder acessory cells, in the form of (B6A)F1 peritoneal or irradiated T cell-depleted spleen cells, or in the form of B10 nonirradiated T cell-depleted spleen cells. These results suggested that (TG)-A--L-specific Ir genes expressed by B cells controlled the ability of these cells to be induced to respond to SRBC by (TG)-A--L-responding T cells, implying that direct contact between the SRBC-binding B cell precursor and the (TG)-A--L-responsive helper T cells was required. Analogous results were obtained for keyhold limpet hemocyanin (KLH)-driven bystander help using KLH-primed F1 T cells restricted to interact with cells on only one of the parental haplotypes by maturing them in parental bone marrow chimeras. It was hypothesized that bystander help was mediated by nonspecific uptake of antigen [(TG)-A--L or KLH] by SRBC-specific b cells and subsequent display of the antigen on the B cell surface in association with Ir of I-region gene products, in a fashion similar to the M0, where it was then recognized by helper T cells. Such an explanation was supported by the observation that high concentrations of antigen were required to elicit bystander help. This hypothesis raises the possibility of B cell processing of antigen bound to its immunoglobulin receptor and subsequent presentation of antigen to helper T cells.

scholarly journals IMMUNOLOGIC REACTIONS TO HAPTENS ON AUTOLOGOUS CARRIERS

1973 ◽  
Vol 137 (2) ◽  
pp. 411-423 ◽  
Author(s):  
John W. Moorhead ◽  
Curla S. Walters ◽  
Henry N. Claman
Keyword(s):  
T Cells ◽  
B Cells ◽  
Aminocaproic Acid ◽  
Single Amino Acid ◽  
Secondary Response ◽  
Spleen Cells ◽  
Activated T Cells ◽  
Thymus Cells

Both thymus-derived (T) and bone marrow-derived (B) lymphocytes participate in the response to a hapten 4-hydroxy-3-iodo-5-nitrophenylacetic acid (NIP), coupled to a nonimmunogenic isologous carrier, mouse gamma globulin (MGG). Spleen cells from mice immunized with NIP-MGG show increased DNA synthesis in vitro when cultured with NIP-MGG. The participation of and requirement for T cells in the response was demonstrated by treating the spleen cells with anti-θ serum. This treatment resulted in a 77% inhibition of the antigen response. Furthermore, adoptively transferred normal thymus cells could be specifically "activated" by NIP-MGG in vivo and they responded secondarily to the antigen in vitro. The active participation of B cells in the secondary response was demonstrated by passing the immune spleen cells through a column coated with polyvalent anti-MGG serum. Column filtration reduced the number of NIP-specific plaque-forming cells and NIP-specific rosette-forming cells (both functions of B cells) and produced a 47% inhibition of the NIP-MGG response. The ability of the cells to respond to phytohemagglutinin (PHA) was not affected by column filtration showing that T cells were not being selectively removed. The participation of B cells in the in vitro NIP-MGG response was also shown by treatment of the spleen cells with antiserum specific for MGG and MGG determinants. B cells were removed by treatment with anti-IgM or polyvalent anti-MGG serum plus complement, resulting in a respective 46 and 49% inhibition of the response to NIP-MGG. (Treatment with anti-IgM serum had no effect on T cells.) The contribution of the hapten NIP to stimulation of T cells was investigated using NIP-MGG-activated thymus cells. These activated T cells responded in vitro very well to the NIP-MGG complex but not to the MGG carrier alone demonstrating the requirement of the hapten for T cell stimulation. The response was also partially inhibited (41%) by incubating the activated cells with NIP coupled to a single amino acid (epsilon-aminocaproic acid) before addition of NIP-MGG. These results demonstrated that T cells recognize the hapten NIP when it is coupled to the isologous carrier MGG.

Antibodies from Donor B Cells Are Not Required for Initiating but Required for Persisting Scleroderma in Chronic Gvhd

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3817-3817
Author(s):  
Hua Jin ◽  
Xiong Ni ◽  
Ruishu Deng ◽  
James Young ◽  
Heather F Johnston ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Cd4 T Cells ◽  
Conflicts Of Interest ◽  
Chronic Gvhd ◽  
Cell Interaction ◽  
Spleen Cells ◽  
Littermate Control ◽  
Inflammatory Status

Abstract We recently reported that in a chronic graft versus host disease (GVHD) model of DBA/2 donor to MHC-matched BALB/c recipient, donor CD4+ T and B cell interaction resulted in not only hyperglobulinemia and glomerulonephritis but also scleroderma (J. Immunol. 2012). It is well known that glomerulonephritis is caused by immune complex deposition. However, the role of antibodies from donor B cells in the pathogenesis of scleroderma remains unclear. To address this question, we generated DBA/2 mice whose B cells have APC function but cannot secrete antibodies by backcrossing IgHµg1 mice from Dr. Rajewsky’s lab (JEM 2007). We observed that, while transplanting T-cell-depleted bone marrow (TCD-BM) and spleen cells from littermate control mice induced proteinuria and scleroderma, transplanting BM and spleen cells from IgHµg1 DBA/2 mice induced no proteinuria, but the recipients developed scleroderma ~35 days after HCT. Interestingly, the scleroderma gradually recovered ~55 days after HCT. 40 days after HCT, scleroderma recipients transplanted with WT spleen cells (Rec-WT) or recipients transplanted with IgHµg1 spleen cells (Rec-IgHµg1) both had high percentage (~12%) of IFN-g+ or IL-17+ CD4+ T cells in the peripheral lymph node (PLN) and skin tissues, as compared to that (~3%) of GVHD-free recipients given TCD-BM alone (Rec-TCD). While Rec-WT had severe reduction of CD4+CD8+ thymocytes, the Rec-IgHµg1 had no reduction of the thymocytes, as compared to that of Rec-TCD. By day 60 after HCT, the Rec-WT with ongoing scleroderma still had ~10% IFN-g+ or IL-17+ CD4+ T cells in the PLN and skin tissues; in contrast, although the Rec-IgHµg1 with reversal of scleroderma still had >10% IFN-g+or IL-17+ CD4+ T cells in the PLN, those cells in the skin had reduced to <2%. This reduction was associated with DC upregulation of B7H1 and T cell upregulation of PD-1. These results suggest that antibodies from B cells are required for maintaining inflammatory status of tissue DCs and persistence of scleroderma in chronic GVHD. (This work was supported by NIH R01 AI066008). Disclosures No relevant conflicts of interest to declare.

scholarly journals The Ets protein Spi-B is expressed exclusively in B cells and T cells during development.

1996 ◽  
Vol 184 (1) ◽  
pp. 203-214 ◽  
Author(s):  
G H Su ◽  
H S Ip ◽  
B S Cobb ◽  
M M Lu ◽  
H M Chen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Expression Patterns ◽  
Lymphoid Cells ◽  
Cell Maturation ◽  
White Pulp ◽  
Ets Family ◽  
T Cell Lines

Spi-B and PU.1 are hematopoietic-specific transcription factors that constitute a subfamily of the Ets family of DNA-binding proteins. Here we show that contrary to previous reports, PU.1 and Spi-B have very different expression patterns. PU.1 is expressed at high levels in B cells, mast cells, megakaryocytes, macrophages, neutrophils, and immature erythroid cells and at lower levels in mature erythrocytes. PU.1 is completely absent from peripheral T cells and most T cell lines based on sensitive RT-PCR assays. In contrast, Spi-B is expressed exclusively in lymphoid cells and can be detected in early fetal thymus and spleen. In situ hybridizations of adult murine tissues demonstrate Spi-B mRNA in the medulla of the thymus, the white pulp of the spleen, and the germinal centers of lymph nodes. Spi-B expression is very abundant in B cells and both Spi-B mRNA and protein are detected in some T cells. In situ hybridization and Northern blot analysis suggest that Spi-B gene expression increases during B cell maturation and decreases during T cell maturation. Gel-retardation experiments show that Spi-B can bind to all putative PU.1 binding sites, but do not reveal any preferred Spi-B binding site. Finally, both PU.1 and Spi-B function as transcriptional activators of the immunoglobulin light-chain enhancer E lambda 2.4 when coexpressed with Pip (PU.1-interaction partner) in NIH-3T3 cells. Taken together, these data suggest that differences in patterns of expression between Spi-B and PU.1 distinguish the function of each protein during development of the immune system.

scholarly journals T-cell regulation of antibody responses: demonstration of allotype-specific helper T cells and their specific removal by suppressor T cells.

1976 ◽  
Vol 144 (2) ◽  
pp. 330-344 ◽  
Author(s):  
L A Herzenberg ◽  
K Okumura ◽  
H Cantor ◽  
V L Sato ◽  
F W Shen ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Antibody Responses ◽  
Helper T Cells ◽  
Spleen Cells ◽  
Suppressor T Cells ◽  
Effector Cells ◽  
T Cell Regulation ◽  
The Difference

Allotype suppressor T cells (Ts) generated in SJL X BALB/c mice specifically suppress production of antibodies marked with the Ig-1a allotype. The studies presented here show that allotypes Ts suppress by specifically removing helper T cell (Th) activity required to facilitate differentiation and expansion of B cells to Ig-1b antibody-forming cells. We show first that Ts and Th belong to different T-cell subclasses as defined by Ly surface antigens. Ts are Ly2+Lyl- and thus belong to the same subclass as cytotoxic precursor and effector cells; Th are Lyl+Ly2- cells and thus belong to the subclass containing cells which can exert helper functions and initiate delayed hypersensitivity reactions. Placing these cells in these two subclasses shows that Th are different from Ts and suggests that they play different roles in regulating antibody responses. The difference in these roles is defined by the evidence presented here showing that Ts attack Th and regulate the antibody response by specifically regulating the availability of Th activity. We show that in allotype suppressed mice, Ts which suppress Ig-1b antibody production have completely removed the Th activity of helping Ig-1b cells without impairing Th activity which helps other IgB B cells. These findings imply the existence of allotype-specific Th for Ig-1b cells (Ig-1b Th). We directly establish that Ig-1b cells require such help by showing that carrier-primed spleen cells from Iga/Iga congenic hybrids help Ig-1a B cells from hapten-primed Igb/Iga donors but do not help Ig-1b B cells from the same donor in the same adoptive recipient.

scholarly journals CELL-TO-CELL INTERACTION IN THE IMMUNE RESPONSE

1974 ◽  
Vol 140 (1) ◽  
pp. 199-217 ◽  
Author(s):  
A. Basten ◽  
J. F. A. P. Miller ◽  
J. Sprent ◽  
C. Cheers
Keyword(s):  
T Cells ◽  
B Cells ◽  
Inhibitory Effect ◽  
Immunological Tolerance ◽  
Primary Response ◽  
Secondary Response ◽  
Thoracic Duct Lymph ◽  
Normal Numbers ◽  
Spleen Cells ◽  
Suppressor Effect

Specific immunological tolerance was induced in CBA mice by a single injection of deaggregated fowl immunoglobulin G (FγG). The unresponsive state was stable on adoptive transfer and irreversible by pretreatment of tolerant cells with trypsin. Tolerant spleen cells could suppress the response of normal syngeneic recipients. They also suppressed the adoptive primary response of spleen cells to FγG in irradiated hosts. The inhibitory effect was on the indirect (7S) plaque-forming cell (PFC) response. Incubation of the tolerant cell population with anti-θ serum and complement reversed the suppressor effect. Furthermore, the addition of purified T cells from normal donors restored the capacity of the anti-θ serum-treated tolerant cells to transfer an adoptive response to FγG. The existence of FγG-reactive B cells was supported by the demonstration of normal numbers of antigen-binding cells in the spleen and thoracic duct lymph from tolerant animals. Moreover, the formation of caps by these cells implied that they could bind antigen normally. These experiments provided direct evidence for the existence of suppressor T cells in the tolerant population. Further evidence was derived from examination of the effect of antigen "suicide". Tolerant spleen cells were treated with radioactive FγG under conditions known to abrogate T-cell helper function. When these cells were transferred together with normal spleen cells into irradiated hosts, suppression of the primary adoptive response to FγG was no longer observed. Inhibition of an adoptive secondary response to FγG was obtained by transferring tolerant spleen cells with primed B cells provided high doses of tolerant cells were used. By contrast low doses exerted a helper rather than a suppressor effect in this system.

scholarly journals CELL-TO-CELL INTERACTION IN THE IMMUNE RESPONSE

1971 ◽  
Vol 134 (5) ◽  
pp. 1266-1284 ◽  
Author(s):  
J. F. A. P. Miller ◽  
J. Sprent ◽  
A. Basten ◽  
N. L. Warner ◽  
J. C. S. Breitner ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Antibody Response ◽  
Cell Types ◽  
Cell Interaction ◽  
Antibody Responses ◽  
Secondary Response ◽  
Spleen Cells ◽  
Control Antigen

Experiments were designed to test the possibility that thymus-derived (T) cells cooperate with nonthymus derived (B) cells in antibody responses by acting as passive carriers of antigen. Thoracic duct lymphocytes (TDL) from fowl γG-tolerant mice were incubated in vitro with fowl anti-mouse lymphocyte globulin (FALG), which was shown not to be immunosuppressive in mice. On transfer into adult thymectomized, irradiated, and marrow protected (TxBM) hosts together with a control antigen, horse RBC, a response to horse RBC but not to fowl γG was obtained. By contrast, TxBM recipients of nontolerant, FALG-coated TDL responded to both antigens and the antibody-forming cells were shown to be derived from the host, not from the injected TDL. These findings suggested that, under the conditions of the experiment, triggering of unprimed B cells in the spleens of TxBM hosts was not achieved with antigen-coated tolerant lymphocytes. Another model utilized the ability of B cells to bind antibody-antigen complexes. Spleen cells from TxBM mice, incubated in vitro with anti-fowl γG-fowl γG·NIP, were injected with or without normal TDL (a source of T cells) into irradiated hosts. Only mice given both cell types could produce an anti-NIP antibody response. In a further experiment, spleen cells from HGG·NIP-primed mice were injected together with NIP-coated B cells (prepared as above) into irradiated hosts. A substantial anti-NIP antibody response occurred. If, however, the T cells in the spleens of HGG·NIP-primed mice were eliminated by treatment with anti-θ serum and complement, the NIP response was abolished. It was concluded that antigen-coated B cells could not substitute for T cells either in the primary or secondary response. Treatment of T cells from unprimed or primed mice with mitomycin C impaired their capacity to collaborate with B cells on transfer into irradiated hosts. Taken together these findings suggest that before collaboration can take place T cells must be activated by antigen to differentiate and in so doing may produce some factor essential for triggering of B cells.

scholarly journals Dynamic BH3 Profiling As Pharmacodynamic Biomarker for the Activity of BH3 Mimetics

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 6-6
Author(s):  
Rongqing Pan ◽  
Youzhen Wang ◽  
Shumei Qiu ◽  
Jeremy Ryan ◽  
Ensar Halilovic
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Biomedical Research ◽  
Lymphoid Cells ◽  
In Vivo Studies ◽  
Functional Assay ◽  
Bh3 Mimetics ◽  
Apoptotic Signaling

Pharmacodynamic (PD) biomarkers provide information about the pharmacologic effects of a drug on its target, i.e. to assess whether a given agent is engaging its target in the expected manner. There is currently no convenient PD marker for BH3 mimetics. BH3 profiling (BP) is a functional assay developed to measure cells susceptibility to apoptosis and cell's dependence on certain Bcl-2 anti-apoptotic proteins for survival. Dynamic BH3 profiling (DBP) measures the changes of BP signals after drug perturbations. In this study, we studied whether DBP with lymphoid cells can serve as a PD biomarker for the activity of BH3 mimetics. Using DBP and cell lines with defined dependency, we first showed that BH3 mimetics BCL201 (S55746) and S63845 are highly selective for Bcl-2 or Mcl-1, respectively. Next, we treated primary human lymphoid cells with BCL201 and then conducted DBP. We asked if pretreatment with BCL201 altered mitochondrial sensitivity to different BH3 peptides. After treatment with 1 μM BCL201, an amount consistent with achievable levels in vivo, T cell sensitivity to the Mcl-1 selective MS1 peptide, but not Bad peptide or Bfl-1 selective FS1 peptide, increased significantly, from 20% to 51%. These results suggest that BCL201 treatment increases T cell dependency on Mcl-1 and that DBP of T cells with MS1 peptide may serve as a PD marker for BCL201 activity. Similar results were also observed in B cells. Next, we conducted DBP on healthy T cells treated with S63845. T cell mitochondrial sensitivity to MS1 peptide or Bcl-xL selective HRK peptide did not change. However, the treatment increased mitochondrial sensitivity to the Bad and FS1 peptides significantly, boosting the signal from 10.2% to 57.2% and 46.8% respectively, suggesting DBP of T cells with Bad or FS1 peptides can be robust PD markers for S63845 activity. This results also indicate that S63845 treatment augmented T cell dependency on Bcl-2 and Bfl-1 for survival. Currently, we are investigating whether DBP can be used as a PD biomarker for in vivo studies. Our preliminary data suggest that DBP works well across different species (rat, mouse, and human). More importantly, DBP can detect changes in mitochondrial apoptotic signaling of ex vivo rat cells after in vivo treatment with BH3 mimetics. Conclusions: Exposure to BH3 mimetics causes changes in mitochondrial apoptotic signaling of T/B cells which are readily detectable by DBP. DBP with lymphoid cells may provide a robust PD biomarker for clinical trials testing BH3 mimetics, either as monotherapy or in combination with other agents. Disclosures Wang: Novartis Institutes for Biomedical Research: Current Employment. Qiu:Novartis Institutes for Biomedical Research: Current Employment. Halilovic:Novartis Institutes for Biomedical Research: Current Employment, Current equity holder in publicly-traded company.

scholarly journals A RECEPTOR FOR ANTIBODY ON B LYMPHOCYTES

1972 ◽  
Vol 135 (3) ◽  
pp. 610-626 ◽  
Author(s):  
A. Basten ◽  
J. F. A. P. Miller ◽  
J. Sprent ◽  
J. Pye
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Lymphocytes ◽  
Antibody Binding ◽  
Lymphoid Cells ◽  
Secondary Response ◽  
Target Cells ◽  
Binding Studies ◽  
Cell Content

Evidence is presented for the existence on all B lymphocytes, but not on T lymphocytes, of a membrane-associated receptor for antibody. The receptor was detected by a radioautographic technique in which lymphoid cells were incubated with antibody followed by the corresponding radioiodinated antigen. The ease with which antibody eluted during washing indicated that the bond between antibody and cell was weak. The formation of an antibody-antigen complex on the cell surface, however, stabilized the bond and permitted accurate quantitation of cells with adherent antibody. The ability of several combinations of antibody and antigen to adhere to the cells demonstrated the nonspecificity of the phenomenon and emphasized the need for care in interpretation of antigen-binding studies particularly when immune cells are being used. The identity of antibody-binding lymphocytes was established by two different approaches. In the first, mouse lymphocyte populations greatly enriched for either T cells or B cells were examined. Their T cell content was assessed by means of well-established markers such as the θ C3H isoantigen. When this was compared with the number of antibody-binding cells, an inverse relationship was obtained in each instance; thus almost all thoracic duct cells from athymic mice labeled with an immune complex although none were θ positive. The striking reduction in antibody-binding cells observed in bursectomized chickens provided a second and independent line of evidence suggesting that B cells, not T cells, bind antibody. The ability of B cells from primed animals to bind antibody in vivo made it important to test whether this phenomenon was related to the carriage of immunological memory. No correlation was, however, found between membrane-bound antibody and memory. It was proposed that the existence of a receptor of this kind may provide a rational explanation for antibody-dependent killing of target cells and may prove of importance in antigen concentration particularly during the secondary response.

A Profile of 648 Signaling Network Events Identifies Cell Subsets with Diverse, Abnormal Responses to Lymphocyte Stimuli within Follicular Lymphoma Tumors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 356-356 ◽  
Author(s):  
Jonathan M. Irish ◽  
Faye Y. Hsu ◽  
Jeff P. Sharman ◽  
Roch Houot ◽  
Joshua D. Brody ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Cell Survival ◽  
Signaling Network ◽  
Lymphoid Cells ◽  
Network Activity ◽  
T Cell Subsets

Abstract Signal transduction plays a key role in cell survival, and changes to signaling are frequently implicated in tumor initiation and progression. We sought to identify abnormal variation in signaling network activity within primary tumor samples obtained prior to treatment from patients with follicular lymphoma (FL). We previously showed that altered B cell receptor (BCR) signaling distinguishes tumor B cells from the non-malignant host B cells in FL tumors. Here we extend this approach and use flow cytometry to measure 648 signaling events in live lymphoid cells from more than 25 lymphoma specimens and healthy controls. We combined 9 previously identified BCR stimulation conditions with inputs from CD40, interleukin 4, interferons (IFNs), and more than 10 other environmental cues that govern the development and activity of lymphocytes. Fluorescent cell barcoding allowed simultaneous staining and analysis of phospho-protein activation under all 27 stimulation conditions within a single tube. The activation of key phospho-protein nodes throughout lymphocyte signaling networks, including Syk, Erk1/2, Btk, Src family kinases, cCbl, p38, NFkB, Akt, Stat1, Stat3, Stat6, and Stat5, was measured under each of the 27 stimulation conditions. Measurements of phospho-protein responses to stimulation were combined with detection of the Bcl-2 oncogene, B and T cell lineage markers in each cell. This panel allowed us to characterize signaling in the heterogeneous cell subsets found within each patient’s tumor sample. Tumor B cells, host tumor infiltrating T cells, non-malignant B cells were all distinguished by contrasting signaling profiles. In some cases, subsets of tumor B cells with differences in signaling network topology were observed within the tumor B cell population. This result suggests that signaling can distinguish between tumor sub-clones and could be used to measure tumor heterogeneity. As previously reported, little variation in signaling was observed among healthy peripheral blood B and T cell samples from different individuals. Abnormally low host T cell signaling was commonly observed within the tumor infiltrating T cells infiltrating FL tumors. Further analysis of tumor T cell subsets indicated that a high proportion of infiltrating T cells expressed CD4 and FoxP3. Taken together, these results support the hypothesis that FL tumor B cells promote suppressed signaling in the T cells of the patient and may modulate the immune response against the tumor. In FL tumor B cells, BCR and IFN signaling frequently triggered Stat5 phosphorylation, but not Stat1 phosphorylation. These results are consistent with the hypothesis that Stat5 initiates genetic programs that support cancer cell survival and proliferation, whereas Stat1 promotes immunogenicity and cooperates with the p53 tumor suppressor protein. In contrast with healthy B cells, loss of the response to CD40L, altered PKC signaling, and variable responses to BCR crosslinking were all seen in FL tumor B cells. The patterns of abnormal signaling we observed in tumor B cells and tumor infiltrating T cells suggest that measuring the activity of key signaling network nodes can identify targets for therapeutic attention in FL.

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