Characterization of activation of a partially defective B cell subpopulation with immunocompetence restricted to IgM and IgA

A B-cell subpopulation (BM-A cell) responding to an antigen with the production of IgM and IgA plaque-forming cells but not of IgG plaque-forming cells was isolated from neonatally bursectomized chickens and was examined for the mode of activation by B-cell mitogens. The BM-A cells did not elevate both glucose consumption and protein synthesis with the B-cell mitogens, in striking contrast to normal B cells. The BM-A cells were also not activated by an activator of protein kinase C, phorbol myristate acetate. Both anti-Ig and a calcium ionophore, A23187, however, primed the BM-A cells to increase intracellular free calcium ion as well as normal B cells. From these results it is conceived that the lack of protein kinase C activation may be responsible for the failure of activation of BM-A cells.     

Role of adhesive lymphoid cell subpopulation in areactivity to hepatoma 22a]

The role of adhesive fraction of T lymphocytes in nonreactivity of mice to hepatoma 22a was studied. It was shown that the removal of the adhesive fraction from the spleen suspension enriched with T lymphocytes promotes intensification of cell immunity in tumor-tolerant mice.     

Dynamics of immune cell recruitment during West Nile encephalitis and identification of a new CD19+B220-BST-2+ leukocyte population

West Nile virus (WNV) is an emerging neurotropic flavivirus. We investigated the dynamics of immune cell recruitment in peripheral tissues and in the CNS during WNV encephalitis in an immunocompetent mouse model. In the periphery, immune cell expansion can successfully limit viremia and lymphoid tissue infection. However, viral clearance in the periphery is too late to prevent viral invasion of the CNS. In the CNS, innate immune cells, including microglia/macrophages, NK cells, and plasmacytoid dendritic cells, greatly expand as the virus invades the brain, whereas B and T cells are recruited after viral invasion, and fail to control the spread of the virus. Thus, the onset of WNV encephalitis was correlated both with CNS viral infection and with a large local increase of innate immune cells. Interestingly, we identify a new immune cell type: CD19(+)B220(-) BST-2(+), which we name G8-ICs. These cells appear during peripheral infection and enter the CNS. G8-ICs express high levels of MHC class II, stain for viral Ag, and are localized in the paracortical zone of lymph nodes, strongly suggesting they are previously unidentified APCs that appear in response to viral infection.     

Isolation, modulatory functions on murine B cell development and antigen-specific immune responses of BP11, a novel peptide from the chicken bursa of Fabricius

The bursa of Fabricius (BF) is the central humoral immune organ unique to birds which plays important roles in B lymphocyte differentiation. Here, a new bursal peptide (BP11) with the amino acid sequence DVAGKLPDNRT was identified and characterized from BF. It was proved that BP11 promoted CFU pre-B formation, and regulated B cell differentiation, including increase the percentage of immature and mature B cells in BM cells co-cultured with IL-7. BP11 also exerted immunomodulatory function on antigen-specific immune responses in BALB/c mice immunized with inactivated influence virus (AIV, H9N2 subtype) vaccine, including enhancing AIV-specific antibody and cytokine production. Furthermore, it was noteworthy that BP11 stimulated antibody productions and potentiates the Th1 and Th2-type immune responses in dose-dependent manner in chicken. These results suggested that BP11 might be highly relevant for the development of avian immune system.     

Characterization of a phenotypically distinct subpopulation of Leu-2+ cells that suppresses T cell proliferative responses

Two new monoclonal antibodies (termed 2D2 and D12) have been used to identify and to analyze phenotypically distinct subpopulations of human T cells. The 2D2 antibody recognized an antigenic determinant closely related, if not identical, to that reactive with the anti-Leu-2 monoclonal antibody. The D12 antibody reacted with a variety of cell types, which included a subpopulation of Leu-2+ (2D2+) T cells. These antibodies were used to isolate four phenotypically distinct T cell populations by sequential cell sorter techniques. Functional analyses demonstrated that the 2D2+D12+ subset was unique in its ability to suppress the antigen-induced proliferation of T cells. These cells also suppressed the proliferative responses of other T cell subsets stimulated with mitogens. Pretreatment of 2D2+D12+ T cells with mitomycin C before culture abrogated the suppressor cell activity of these cells. We propose that the cells within the Leu-2+ cytotoxic/suppressor T cell subpopulation that suppress T cell proliferation are phenotypically distinct and express the 2D2+D12+ membrane antigenic phenotype.     

In vitro cell culture model of human nasal-associated lymphoid tissue (NALT) to evaluate the humoral immune response to SARS-CoV-2 spike proteins

To date, coronavirus disease 2019 (COVID-19) continues to be considered a pandemic worldwide, with a mild to severe disease presentation that is sometimes associated with serious complications that are concerning to global health authorities. Scientists are working hard to understand the pathogenicity of this novel virus, and a great deal of attention and effort has been focused on identifying therapeutics and vaccines to control this pandemic.MethodsThis study used tonsils removed from twelve patients who underwent an elective tonsillectomy in the ear, nose, and throat (ENT) department at Saudi Germany Hospital, Madinah, Saudi Arabia. Tonsillar mononuclear cells (MNCs) were separated and co-cultured in RPMI complete medium in the presence and absence of viral spike (S) proteins (the full-length S, S1 subunit, and S2 subunit proteins). Enzyme-linked immunosorbent assay (ELISA) was used to measure secreted antibody concentrations following stimulation.ResultsThe in vitro human nasal-associated lymphoid tissue (NALT) cell culture model was successfully used to evaluate the humoral immune response against SARS-CoV-2- S protein. Significant (p < 0.0001, n = 12) levels of specific, anti-S IgG, IgM, and IgA antibody responses were detected in cells culture supernatanat folloeing stimulation with the full-length S protein compared with unstimulated cells. In contrast, S1 and S2 subunit proteins alone failed to induce a mucosal humoral immune response following tonsillar MNC stimulation.ConclusionWe demonstrated a successful human NALT in vitro cell culture model that was used to study the mucosal humoral immune response to the SARS-CoV-2 S protein. This model could be advantageous for the in-depth study of cellular immune responses to the S protein and other viral antigens, such as nucleocapsid and matrix antigen. The S protein appears to be the important viral protein that may be able to mimic the natural infection process intranasally and should be studied as a component of a candidate vaccine.     

Development and maintenance of a B220- memory B cell compartment

We have recently demonstrated that a novel somatically mutated B220(-) memory B cell subset rapidly dominates the secondary immune response to (4-hydroxy-3-nitrophenyl) acetyl (NP). Upon adoptive transfer with Ag, B220(+)NP(+) memory B cells produce large numbers of B220(-)NP(+) B cells that can rapidly differentiate into plasma cells. Therefore, it is not clear whether the novel B220(-) memory compartment is a consequence of secondary Ag challenge or whether it develops as a stable memory subset after initial Ag challenge. In this study, we demonstrate the gradual emergence of B220(-)NP(+) B cells in the spleen to maximal numbers 3 wk after initial Ag exposure. Like their B220(+) counterparts, the B220(-) B cells initially appear unmutated at days 5-7; however, the majority rapidly accumulate affinity increasing mutations by days 9-14 of the primary immune response. More extensive cell surface phenotype (GL7(-)BLA-1(-)CD24(-)CD43(+)) argues strongly against germinal center localization and direct analysis in situ places a cohort of B220(-)CD11b(+)NP(+) B cells in the red pulp of the spleen and not in the MZs. These data provide direct evidence for the development of B220(-) memory B cells as a unique cellular consequence of primary Ag exposure. The cellular dynamics and molecular attributes of these unique memory B cells suggest they are distinct cellular products of the germinal center reaction in the primary response and are maintained long-term in the spleen and bone marrow.     

Liver-derived DEC205+B220+CD19- dendritic cells regulate T cell responses

Leukocytes resident in the liver may play a role in immune responses. We describe a cell population propagated from mouse liver nonparenchymal cells in IL-3 and anti-CD40 mAb that exhibits a distinct surface immunophenotype and function in directing differentiation of naive allogeneic T cells. After culture, such cells are DEC-205(bright)B220+CD11c-CD19-, and negative for T (CD3, CD4, CD8alpha), NK (NK 1.1) cell markers, and myeloid Ags (CD11b, CD13, CD14). These liver-derived DEC205+B220+ CD19- cells have a morphology and migratory capacity similar to dendritic cells. Interestingly, they possess Ig gene rearrangements, but lack Ig molecule expression on the cell surface. They induce low thymidine uptake of allogeneic T cells in MLR due to extensive apoptosis of activated T cells. T cell proliferation is restored by addition of the common caspase inhibitor peptide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-fmk). T cells stimulated by liver-derived DEC205+B220+D19- cells release both IL-10 and IFN-gamma, small amounts of TGF-beta, and no IL-2 or IL-4, a cytokine profile resembling T regulatory type 1 cells. Expression of IL-10 and IFN-gamma, but not bioactive IL-12 in liver DEC205+B220+CD19- cells was demonstrated by RNase protection assay. In vivo administration of liver DEC205+B220+CD19- cells significantly prolonged the survival of vascularized cardiac allografts in an alloantigen-specific manner.     

Supplementation with selenium and human immune cell functions

Biological Trace Element Research - This study examined the effect of dietary (200 μg/d for 8 wk) supplementation with selenium (as sodium selenite) on the ability of human peripheral blood...     

Characterization of a B cell helper factor(s) derived from CD5+ B cell hybridomas.

Work from our laboratory suggests that the selective advantage of frequently autoreactive CD5+ B cells is to provide activation signals to CD5- antigen-specific B cells. This hypothesis is supported by the observation that supernatants from CD5+ B cell hybridomas replace CD5+ B cell populations in helping idiotypic B cell subsets respond to antigen plus anti-idiotype antibody. The present study was designed to initiate the characterization of CD5+ B hybridoma-derived helper factor(s) (BHF) and to compare BHF to previously described cytokines. Elution of BHF from a lectin column enabled significant enrichment of the apparently glycosylated helper factor(s) from serum-free hybridoma supernatant. Gel filtration of this enriched activity revealed two significant peaks of helper activity, one at approximately 19-22 kDa and a second at 29-32 kDa. BHF activity in each fraction was sensitive to protease treatment. To determine if some previously described cytokines of approximately the same molecular weights were responsible for BHF activity, BHF fractions were tested for cytokine activity in respective bioassays. At least 2000 units of BHF did not contain detectable levels of IL-1, IL-2, IL-3, IL-4, IL-6, GM-CSF, G-CSF, or IFN-gamma activity. Furthermore, three hybridomas which produced BHF did not transcribe detectable levels of mRNAs specific for IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, GM-CSF, or IFN-gamma. The results suggest that CD5+ B cell hybridomas produce a lymphokine(s) distinct from cytokines commonly associated with B cell activation. The potential roles of this lymphokine in immunity and disease are discussed.     

IL-15 inhibits pre-B cell proliferation by selectively expanding Mac-1+B220+ NK cells

Natural killer (NK) cells are the cells critical for inhibition of repopulation of allogenic bone marrow cells. However, it is not well known if NK cells affect autologous lymphopoiesis. Here, we observed that NK cells could inhibit pre-B cell proliferation in vitro driven by interleukin (IL)-7 in a manner dependent on IL-15. Interestingly, the great majority of expanding NK cells were Mac-1⁺B220⁺, a recently identified potent interferon (IFN)-γ producer. Indeed, IFN-γ was produced in those cultures, and pre-B cells lacking IFN-γ receptors, but not those lacking type I IFN receptors, were resistant to such an inhibition. Furthermore, even NK cells from mice lacking β2-microglobulin, which were known to be functionally dampened, inhibited pre-B cell proliferation as well. Thus, activated NK cells, which were expanded selectively by IL-15, could potentially regulate B lymphopoiesis through IFN-γ beyond the selection imposed upon self-recognition.     

Reticulum cells in the ontogeny of nasal-associated lymphoid tissue (NALT) in the rat

This study concerns the ontogeny of reticulum cells (RC) in the nasal-associated lymphoid tissue (NALT) of Wistar and Brown-Norway rats. A panel of monoclonal antibodies (mAb) directed against RC in peripheral lymphoid organs (antibodies ED10ED15) was used, together with a recently developed antibody ED17, which recognizes macrophages and Langerhans cells. Early in embryogenesis, staining with common connective tissue markers, ED14 and ED15, was found. ED17-positive cells were present before cells positive to ED1, a pan-macrophage marker, or Ia glycoproteins were observed. The first differentiation of reticulum was seen at the day of birth, when ED10 recognized a distinct area in the nasal mucosa. The first T-lymphocytes were found at the same time. Two days after birth, B-cells and ED11-positive cells were present in the NALT area. Fourteen days after birth, T- and B-cell compartments were recognizable. ED10 was found predominantly in the T-cell area and ED11 was mainly confined to the B-cell compartment. We conclude that the development of the NALT is closely accompanied by the phenotypic specialization of the reticulum. This suggests that the reticulum plays an important role in the compartmentalization of NALT tissue and in the retention of lymphocyte subsets within these compartments.     

Morphologic and phenotypic features of the subpopulation of Leu-2+ cells that suppresses B cell differentiation

The Leu-2 antigen is expressed on a subpopulation of human T cells that perform suppressor and cytotoxic functions. In addition, this antigen is also present on a portion of cells with morphologic characteristics of granular lymphocytes. Although both Leu-2+ cells and granular lymphocytes have been shown to suppress B cell differentiation, the interrelationship of these two suppressor populations has not previously been fully characterized. We recently produced a monoclonal antibody, termed D12 (anti-Leu-15), which reacts with a variety of cell types, including a subpopulation of Leu-2+ cells. Previous studies have indicated that the Leu-2+ cells that suppress T cell proliferative responses express the Leu-2+15+ phenotype, whereas the precursor and effector cytotoxic T cells that recognize class I major histocompatibility antigens are Leu-2+15- lymphocytes. For this report, we used the anti-Leu-2 and anti-Leu-15 monoclonal antibodies and fluorescence-activated cell sorter techniques to characterize the E+ cells that suppress PWM-induced B cell differentiation. These studies indicate that the vast majority of Leu-2+ cells that suppress this T cell-dependent B cell response have the Leu-2+15+ phenotype. Furthermore, when the morphologic and cytochemical characteristics of these Leu-2+15+ cells were studied, virtually all of these cells were granular lymphocytes. Most of the Leu-2+15+ suppressor cells co-expressed the HNK-1 (Leu-7) antigen, which is detected only on granular lymphocytes. In contrast, virtually none of the Leu-2+15+ granular lymphocytes expressed Fc receptors for IgG molecules. These data indicate that the Leu-2+ cells that suppress PWM-induced B cell differentiation are Leu-2+15+ (and predominantly Leu-7+) granular lymphocytes that do not express Fc receptors. The implications of these observations concerning the relationship of human Leu-2+ suppressor cells to murine Ly-2+ cells and the lineage of granular lymphocytes are discussed.     

Characterization of multiple mRNA species of simian immunodeficiency virus from macaques in a CD4+ lymphoid cell line.

Cytoplasmic poly(A)+ RNA was isolated from CEMX721.174 cells 5 to 10 days after infection with molecularly cloned simian immunodeficiency virus SIVmac239. Expression of SIV RNA was analyzed by Northern (RNA) blot hybridization and by sequencing of cDNA clones. As expected, a splice donor site was demonstrated in the untranslated leader sequence outside the left long terminal repeat. The region between pol and env was found to contain at least two splice donor and six splice acceptor sites. Splice acceptor and donor sites in the intergenic region were suitably positioned for expression of vpx, vpr, tat, and rev. Splice acceptor sites at nucleotides 8802 and 8805 were demonstrated in singly and doubly spliced RNAs with the potential of expressing nef and the second exons of tat and rev. Our results demonstrate a complex pattern of alternative splicing of SIV mRNAs. The results are very similar to what has been observed in human immunodeficiency virus type 1-infected cells, suggesting that both human and simian immunodeficiency viruses are subject to multiple levels of regulation.     

Effect of a single administration of testosterone on the immune response and lymphoid tissues in mice.

Female mice were given a single intraperitoneal injection of testosterone immediately after irradiation and marrow reconstitution. Thirty days later testosterone had no suppressive effect on the recovery of thymus and spleen weights. Testosterone had no effect on the graft-versus-host reaction. Testosterone had no influence on the survival of the skin homografts. However, the plaque-forming cell response to sheep erythrocytes in the spleen was dramatically suppressed by testosterone. Histological observations revealed marked inhibition of lymphoid regeneration selectively in the thymus-independent areas of the peripheral lymphoid tissues. These results suggest that testosterone would act mainly on the differentiation of stem cells toward the population of bone marrow-derived B lymphocytes. The immune response to sheep erythrocytes was restored completely 90 days after testosterone administration. Testosterone given to normal adult mice can also have suppressive activity on the immune system 30 days after a single intraperitoneal injection.     

Leu-1+ (CD5+) B cells. A major lymphoid subpopulation in human fetal spleen: phenotypic and functional studies

Examination of the cell surface phenotype of fetal splenic lymphocytes demonstrated a major, novel subpopulation of B cells that co-express Leu-1 (CD5) in addition to B cell differentiation antigens (Leu-1+ B cells). These cells are similar to some conventional B cells in that they express HLA-DR, Leu-12, and B1, as well as both immunoglobulin (Ig) M and IgD. They comprise 40 to 60% of total splenic B cells in the fetus but are infrequent in fetal liver and adult spleen. Fetal Leu-1+ B cells do not respond to pokeweed mitogen with either proliferation or Ig secretion, and in contrast to the murine counterpart, Ly-1 B cells, they do not constitutively produce Ig. Leu-1+ B cells were incapable of augmenting Ig production of Leu-1- B cells when suboptimal numbers of T cells were present; however, they did require the presence of T cells to secrete antibody. They do not cap either the CD5 protein or surface Ig. These cells are a unique subpopulation of fetal splenic B cells that do not function as conventional B cells. Their role in the humoral immune response is unknown. They may represent the normal stage of B cell development, which is reflected in the phenotype of B cell CLL cells.