Local radiation therapy of B16 melanoma tumors increases the generation of tumor antigen-specific effector cells that traffic to the tumor

Immunotherapy of cancer is attractive because of its potential for specificity and limited side effects. The efficacy of this approach may be improved by providing adjuvant signals and an inflammatory environment for immune cell activation. We evaluated antitumor immune responses in mice after treatment of OVA-expressing B16-F0 tumors with single (15 Gy) or fractionated (5 x 3 Gy) doses of localized ionizing radiation. Irradiated mice had cells with greater capability to present tumor Ags and specific T cells that secreted IFN-gamma upon peptide stimulation within tumor-draining lymph nodes than nonirradiated mice. Immune activation in tumor-draining lymph nodes correlated with an increase in the number of CD45(+) cells infiltrating single dose irradiated tumors compared with nonirradiated mice. Similarly, irradiated mice had increased numbers of tumor-infiltrating lymphocytes that secreted IFN-gamma and lysed tumor cell targets. Peptide-specific IFN-gamma responses were directed against both the class I and class II MHC-restricted OVA peptides OVA(257-264) and OVA(323-339), respectively, as well as the endogenous class I MHC-restricted B16 tumor peptide tyrosinase-related protein 2(180-188). Adoptive transfer studies indicated that the increased numbers of tumor Ag-specific immune cells within irradiated tumors were most likely due to enhanced trafficking of these cells to the tumor site. Together these results suggest that localized radiation can increase both the generation of antitumor immune effector cells and their trafficking to the tumor site.     

Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis

Excessive accumulation of smooth-muscle cells (SMCs) has a key role in the pathogenesis of vascular diseases. It has been assumed that SMCs derived from the outer medial layer migrate, proliferate and synthesize extracellular matrix components on the luminal side of the vessel. Although much effort has been devoted to targeting migration and proliferation of medial SMCs, there is no effective therapy that prevents occlusive vascular remodeling. We show here that in models of post-angioplasty restenosis, graft vasculopathy and hyperlipidemia-induced atherosclerosis, bone-marrow cells give rise to most of the SMCs that contribute to arterial remodeling. Notably, purified hematopoietic stem cells differentiate into SMCs in vitro and in vivo. Our findings indicate that somatic stem cells contribute to pathological remodeling of remote organs, and may provide the basis for the development of new therapeutic strategies for vascular diseases through targeting mobilization, homing, differentiation and proliferation of bone marrow-derived vascular progenitor cells.     

Adoptive transfer of minor histocompatibility antigen-specific T lymphocytes eradicates leukemia cells without causing graft-versus-host disease.

Adoptive transfer of T cells reactive to minor histocompatibility antigens has the unmatched ability to eradicate malignant hematopoietic cells. Unfortunately, its use is hampered by the associated graft-versus-host disease. The critical issue of a possible dissociation of the antileukemic effect and graft-versus-host disease by targeting specific minor histocompatibility antigens remains unresolved because of the unknown nature and number of minor histocompatibility antigens necessary or sufficient to elicit anti-leukemic activity and graft-versus-host disease. We found that injection of T lymphocytes primed against a single major histocompatibility complex class I-restricted immunodominant minor histocompatibility antigen (B6dom1) caused no graft-versus-host disease but produced a curative anti-leukemic response. Avoidance of graft-versus-host disease required that no other host-reactive T cells be co-injected with T cells primed with B6dom1. Here we show that effective and non-toxic immunotherapy of hematologic malignancies can be achieved by targeting a single immunodominant minor histocompatibility antigen.     

Anti-idiotype stimulation of antigen-specific antigen-independent antibody responses in vitro. II. Triggering of B lymphocytes by idiotype plus anti-idiotype in the absence of T lymphocytes

Antibodies specific for the idiotypes of B10 anti-(T,G)-A--L antibodies (anti-id) induced B lymphocytes to secrete anti-(T,G)-A--L antibodies in vitro in the absence of both antigen and T lymphocytes, provided either that the B lymphocytes were previously primed in vivo with (T,G)-A--L or that id specific for (T,G)-A--L was added to the cultures. These antigen- and T lymphocyte-independent responses were antigen specific and appeared not to require accessory cells. The results suggested that B lymphocyte activation occurred via the formation of id-anti-id complexes, and evidence was obtained that this activation involved two separate interactions between the B lymphocytes and the id-anti-id complexes. These studies document a previously undescribed regulatory function of anti-idiotype antibodies.     

Activation of human B lymphocytes. XVII. Synergy between nonspecific and specific signals in the antigen-specific responses of human B cells

The incubation of human peripheral blood lymphocytes (PBL) with the natural killer (NK)-sensitive MOLT-4 cell line results in PBL-target cell conjugate formation by certain lymphocyte subpopulations. Following velocity sedimentation, the PBL depleted of these conjugate-forming subpopulations are markedly diminished in the ability to mediate either antibody-dependent cellular cytotoxicity (ADCC) or NK activity. The immediate testing of highly pure PBL subpopulations isolated from the NK target conjugates does not reveal the expected recovery of augmented ADCC or NK levels. Following in vitro incubation, however, the PBL NK target-binding subpopulations do manifest augmented levels of both NK and ADCC, whereas the depleted PBL continue to display diminished NK and ADCC levels. In addition, the degree of augmented NK and ADCC levels recovered by the NK target-binding PBL subpopulations appears dependent on both the time and the temperature of in vitro incubation. Moreover, the ADCC recovery patterns are identical to those observed for NK activity regardless of the time and temperature of in vitro incubation. These results directly demonstrate that the PBL subpopulations isolated from certain NK target cells are functionally enriched in the ability to mediate from ADCC and NK activity.     

Kinetic analysis of F-actin depolymerization in polymorphonuclear leukocyte lysates indicates that chemoattractant stimulation increases actin filament number without altering the filament length distribution

The rate of filamentous actin (F-actin) depolymerization is proportional to the number of filaments depolarizing and changes in the rate are proportional to changes in filament number. To determine the number and length of actin filaments in polymorphonuclear leukocytes and the change in filament number and length that occurs during the increase in F-actin upon chemoattractant stimulation, the time course of cellular F-actin depolymerization in lysates of control and peptide-stimulated cells was examined. F-actin was quantified by the TRITC-labeled phalloidin staining of pelletable actin. Lysis in 1.2 M KCl and 10 microM DNase I minimized the effects of F-actin binding proteins and G-actin, respectively, on the kinetics of depolymerization. To determine filament number and length from a depolymerization time course, depolymerization kinetics must be limited by the actin monomer dissociation rate. Comparison of time courses of depolymerization in the presence (pointed ends free) or absence (barbed and pointed ends free) of cytochalasin suggested depolymerization occurred from both ends of the filament and that monomer dissociation was rate limiting. Control cells had 1.7 +/- 0.4 x 10(5) filaments with an average length of 0.29 +/- 0.09 microns. Chemo-attractant stimulation for 90 s at room temperature with 0.02 microM N-formylnorleucylleucylphenylalanine caused a twofold increase in F-actin and about a two-fold increase in the total number of actin filaments to 4.0 +/- 0.5 x 10(5) filaments with an average length of 0.27 +/- 0.07 microns. In both cases, most (approximately 80%) of the filaments were quite short (less than or equal to 0.18 micron). The length distributions of actin filaments in stimulated and control cells were similar.     

Activation of antigen-specific CD4+ Th2 cells and B cells in vivo increases norepinephrine release in the spleen and bone marrow

The neurotransmitter norepinephrine (NE) binds to the beta 2-adrenergic receptor (beta 2AR) expressed on various immune cells to influence cell homing, proliferation, and function. Previous reports showed that NE stimulation of the B cell beta 2AR is necessary for the maintenance of an optimal primary and secondary Th2 cell-dependent Ab response in vivo. In the present study we investigated the mechanism by which activation of Ag-specific CD4+ Th2 cells and B cells in vivo by a soluble protein Ag increases NE release in the spleen and bone marrow. Our model system used scid mice that were reconstituted with a clone of keyhole limpet hemocyanin-specific Th2 cells and trinitrophenyl-specific B cells. Following immunization, the rate of NE release in the spleen and bone marrow was determined using [3H]NE turnover analysis. Immunization of reconstituted scid mice with a cognate Ag increased the rate of NE release in the spleen and bone marrow 18-25 h, but not 1-8 h, following immunization. In contrast, immunization of mice with a noncognate Ag had no effect on the rate of NE release at any time. The cognate Ag-induced increase in NE release was partially blocked by ganglionic blockade with chlorisondamine, suggesting a role for both pre- and postganglionic signals in regulating NE release. Thus, activation of Ag-specific Th2 cells and B cells in vivo by a soluble protein Ag increases the rate of NE release and turnover in the spleen and bone marrow 18-25 h after immunization.     

Transfer of memory cells into antigen-pretreated hosts. I. Functional detection of migration sites for antigen-specific B cells.

The distribution of functionally active hapten-specific B memory cells was investigated. Using antigen-pretreated lethally irradiated recipients, a marked accumulation of adoptively transferred B memory cells was demonstrated in lymph nodes containing specific antigen, but not in lymph nodes containing non-cross-reacting hapten conjugates. This difference in responsiveness between lymph nodes containing specific versus those containing nonspecific antigen developed over a period 3–5 days after memory cell transfer. The localization of antigen specific cells was T-cell independent; both carrier-primed T helper cells and specific antigenic challenge, however, were required to trigger the localized B memory cells into antibody production. Specific B memory cell accumulation did not result from an expansion of the antigen-specific cell population due to local proliferation induced by antigen depots in the lymph nodes to challenge. Rather, the results indicated that recirculating B memory cells had progressively accumulated through retention by antigen in the lymph node. These findings suggest that, in the absence of T-cell help and specific antigenic challenge, B memory cells accumulate in lymphoid tissue (follicles) without responding and provide persistent local memory for the humoral immune response.     

The mucosal adjuvant macrophage-activating lipopeptide-2 directly stimulates B lymphocytes via the TLR2 without the need of accessory cells

The macrophage-activating lipopeptide-2 (MALP-2) is an agonist of the TLR heterodimer 2/6, which exhibits potent activity as mucosal adjuvant, promoting strong humoral and cellular responses. Although B cells expressing TLR2/6 are potential targets, very little is known about the effect of MALP-2 on B cells. Studies were performed using total spleen cells or purified B cells from WT mice or animals deficient in TLR2, T cells, B cells, or specific subpopulations of B cells. They demonstrated that MALP-2 promotes a T cell-independent activation and maturation of B cells (mainly follicular but also B-1a and marginal zone B cells) via TLR2. MALP-2 also increased the frequency of IgM- and IgG-secreting cells, but bystander cells were required for IgA secretion. Activated B cells exhibited increased expression of activation markers and ligands that are critical for cross-talk with T cells (CD19, CD25, CD80, CD86, MHC I, MHC II, and CD40). Immunization of mice lacking T cells showed that MALP-2-mediated stimulation of TLR2/6 was unable to circumvent the need of T cell help for efficient Ag-specific B cell activation. Immunization of mice lacking B cells demonstrated that B cells are critical for MALP-2-dependent improvement of T cell responses. The knowledge emerging from this work suggests that MALP-2-mediated activation of B cells through TLR2/6 is critical for adjuvanticity. B cell stimulation by pattern recognition receptors seems to be a basic mechanism that can be exploited to improve the immunogenicity of vaccine formulations.     

Anti-immunoglobulin stimulation of murine lymphocytes. III. Enhancement of the development of responsive B cells by thymic deprivation.

The development of splenic B cells that can be induced to proliferate by soluble anti-immunoglobulin (anti-Ig) reagents requires 7 to 9 months in normal mice. We have found that this age-associated response is enhanced by thymic deprivation. Both neonatally thymectomized LAF₁ mice and thymectomized, lethally irradiated, and bone marrow-restored Balb/c mice respond earlier and more strongly to anti-Ig than their sham controls. Nevertheless, at least 3–4 months are still required after thymectomy before a response can be measured. The earlier and enhanced response to anti-Ig seen in thymectomized animals is not due simply to an increase in the total number of Ig-positive spleen cells. The age-associated response of splenic B cells to anti-Ig we have observed in normal mice may be explained by the “natural” loss of thymic influence that occurs with age.     

Identification of a precursor to phosphatidyl choline-specific B-1 cells suggesting that B-1 cells differentiate from splenic conventional B cells in vivo: cyclosporin A blocks differentiation to B-1

The origin of B-1 cells is controversial. The initial paradigm posited that B-1 and B-2 cells derive from separate lineages. More recently it has been argued that B-1 cells derive from conventional B cells as a result of T-independent Ag activation. To understand B-1 cell differentiation, we have generated Ig transgenic (Tg) mice using the H and L chain genes (VH12 and Vkappa4) of anti-phosphatidyl choline (anti-PtC) B cells. In normal mice anti-PtC B cells segregate to B-1. Segregation is intact in VH12 (6-1) and VH12/Vkappa4 (double) Tg mice that develop large numbers of PtC-specific B cells. However, if B-1 cell differentiation is blocked, anti-PtC B cells in these Tg mice are B-2-like in phenotype, suggesting the existence of an Ag-driven differentiative pathway from B-2 to B-1. In this study, we show that double Tg mice have a population of anti-PtC B cells that have the phenotypic characteristics of both B-2 and B-1 cells and that have the potential to differentiate to B-1 (B-1a and B-1b). Cyclosporin A blocks this differentiation and induces a more B-2-like phenotype in these cells. These findings indicate that these cells are intermediate between B-2 and B-1, further evidence of a B-2 to B-1 differentiative pathway.     

Extreme divergence of a novel wheat thionin generated by a mutational burst specifically affecting the mature protein domain of the precursor.

A new type of neutral thionin (type V), specifically expressed in developing wheat endosperm, has been found to be encoded by a set of single-copy genes located in the long arms of chromosomes 1A, 1B and 1D, within less than 10,000 base-pairs of those corresponding to the highly basic type-I thionins. Divergence between types I and V has occurred through a process of accelerated evolution that has affected the amino acid sequence of the mature thionin but not the precursor domains corresponding to the N-terminal signal peptide and the long C-terminal acidic peptide. This process involved a deletion and a non-synonymous nucleotide substitution rate equal to the synonymous rate in the thionin sequence.     

Hormonal stimulation increases the recruitment of bone marrow-derived myoepithelial cells and periductal fibroblasts into the mammary gland

Recent reports have revealed that bone marrow (BM)-derived cells can be constituents in a number of organs, especially in remodeling tissue. Using bone marrow transplantation (BMT) technique, we found that BM can serve as a source of both myoepithelial cells and periductal fibroblasts in the mammary gland. The numbers of BM-derived myoepithelial cell were 4.8-fold, and those of periductal fibroblast were 2.4-fold higher in the mice when BMT which was performed at the pubertal stage, as compared with BMT was performed at the postpubertal stage. Treatment with estrogen+progesterone pellet increased numbers of BM-derived myoepithelial cells and periductal fibroblasts, to levels 4.5- and 2.6-fold higher than in placebo mice, respectively. In situ hybridization revealed BM-derived periductal fibroblasts expressed insulin-like growth factor I mRNAs that are known to regulate mammary gland. These results suggest that drastic structural change that is induced by hormonal stimulation increased the recruitment of BM-derived myoepithelial cells and periductal fibroblasts to the mammary gland context.     

CD2 facilitates differentiation of CD4 Th cells without affecting Th1/Th2 polarization.

The role of CD2 in murine CD4 helper T cell differentiation and polarization was examined using TCR-Cyt-5CC7-I transgenic recombination activating gene-2-/- H-2(a) mice on CD2+/+ or CD2-/- backgrounds. In the absence of CD2, thymic development was abnormal as judged by reduction in the steady state number of total, double-positive, and CD4 single-positive (SP) thymocytes, as well as a defect in their restorative dynamics after peptide-induced negative selection in vivo. In addition, in CD2-/- animals, lymph node CD4 SP T cells manifest a 10- to 100-fold attenuated activation response to cytochrome c (CytC) agonist peptides as judged by induction of CD25 and CD69 cell surface expression or [(3)H]TdR incorporation; differences in the magnitude of responsiveness and requisite molar peptide concentrations were even greater for altered peptide ligands. Although the presence or absence of CD2 did not impact the final Th1 or Th2 polarization outcome, CD2 expression reduced the CytC peptide concentration threshold necessary to facilitate both Th1 and Th2 differentiation. In vivo administration of CytC peptide to CD2-/- animals yielded an impaired CD4 SP T cell effector/memory phenotype compared with similarly treated CD2+/+ mice. Analysis of TCR-Cyt-5CC7-I human CD2 double-transgenic mice similarly failed to reveal a preferential Th1 vs Th2 polarization. Collectively, these results indicate that CD2 is important for the efficient development of CD4 SP thymocytes and TCR-dependent activation of mature CD4 lymph node T cells, but does not direct a particular helper T cell subset polarity.