Rat Popliteal Lymph Nodes Research Articles

Non-Invasive Detection, Precise Localization, and Perioperative Navigation of In Vivo Deep Lesions Using Transmission Raman Spectroscopy.

Non-invasive detection and precise localization of deep lesions have attracted significant attention for both fundamental and clinical studies. Optical modality techniques are promising with high sensitivity and molecular specificity, but are limited by shallow tissue penetration and the failure to accurately determine lesion depth. Herethe authors report in vivo ratiometric surface-enhanced transmission Raman spectroscopy (SETRS) for non-invasive localization and perioperative surgery navigation of deep sentinel lymph nodes in live rats. The SETRS system uses ultrabright surface-enhanced Raman spectroscopy (SERS) nanoparticles with a low detection limit of 10 pM and a home-built photosafe transmission Raman spectroscopysetup. The ratiometric SETRS strategy is proposed based on the ratio of multiple Raman spectral peaks for obtaining lesion depth. Via this strategy, the depth of the phantom lesions in ex vivo rat tissues is precisely determined with a mean-absolute-percentage-error of 11.8%, and the accurate localization of a 6-mm-deep rat popliteal lymph node is achieved. The feasibility of ratiometric SETRS allows the successful perioperative navigation of in vivo lymph node biopsy surgery in live rats under clinically safe laser irradiance. This study represents a significant step toward the clinical translation of TRS techniques, providing new insights for the design and implementation of in vivo SERS applications.

Paired-Agent Fluorescence Molecular Imaging of Sentinel Lymph Nodes Using Indocyanine Green as a Control Agent for Antibody-Based Targeted Agents.

Purpose Paired-agent molecular imaging methods, which employ coadministration of an untargeted, “control” imaging agent with a targeted agent to correct for nonspecific uptake, have been demonstrated to detect 200 cancer cells in a mouse model of metastatic breast cancer. This study demonstrates that indocyanine green (ICG), which is approved for human use, is an ideal control agent for future paired-agent studies to facilitate eventual clinical translation. Methods The kinetics of ICG were compared with a known ideal control imaging agent, IRDye-700DX-labeled antibody in both healthy and metastatic rat popliteal lymph nodes after coadministration, intradermally in the footpad. Results The kinetics of ICG and antibody-based imaging agent in tumor-free rat lymph nodes demonstrated a strong correlation with each other (r = 0.98, p < 0.001) with a measured binding potential of −0.102 ± 0.03 at 20 min postagent injection, while the kinetics of ICG and targeted imaging agent shows significant separation in the metastatic lymph nodes. Conclusion This study indicated a potential for microscopic sensitivity to cancer spread in sentinel lymph nodes using ICG as a control agent for antibody-based molecular imaging assays.

Assessment of sensitization potential of monoterpenes using the rat popliteal lymph node assay

The popliteal lymph node assay (PLNA) has been proposed as a screening test for detecting chemicals with potential of inducing allergic and auto-immune-like reactions in humans. In the present study, we used the rat PLNA to evaluate the immuno-sensitizing potential of 10 monoterpenes found in the essential oils of a variety of aromatic, edible and medicinal plants. The primary or direct PLNA was performed with the monoterpenes, and chlorpromazine (CPZ) and barbital were used as positive and negative controls, respectively. Female, 7–8 week-old Wistar rats were injected subcutaneously (50 μL) with the test substance (0.5, 2.5 or 5 mg) into the right hind footpad while the contralateral footpad was injected with the vehicle (DMSO) alone. Weight (WI) and cellularity (CI) indices for draining PLNs were determined 7 days after treatment. PLNA was positive (WI ⩾ 2 and CI ⩾ 5) for CPZ, citral, α-terpinene, β-myrcene and (−)-α-pinene, and negative for barbital, DMSO, (−)-menthol, 1,8-cineole, (±) citronellal, (+)-limonene, (±) camphor and terpineol. A secondary PLNA, a T-cell priming test, was carried out with the four substances that had been positive in the primary assay. Six weeks after being locally primed with 5 mg/paw, rats were sc injected into the same footpad with a dose (0.5 mg/paw) of the substance that had been previously found to be insufficient to cause a positive response. WI and CI were then calculated 4 and 7 days after the second injection. CPZ was also positive in the secondary assay thereby confirming that it is a sensitizing agent. Citral, α-terpinene, β-myrcene and (−)-α-pinene, however, were negative in the secondary assay. In summary, citral, α-terpinene, β-myrcene and (−)-α-pinene induced a clear immuno-stimulatory response due to their irritant properties but no monoterpene proved to be a sensitizing agent in the PLNA.

Basal induction of cytochrome P-450 1A1/1A2 in cells of the liver, small intestinal villi, and lymph nodes in rats

Rat liver, small intestinal wall, mesenteric, mediastinal, and popliteal lymph nodes of rats were studied by indirect immunoperoxidase method with monoclonal antibodies to cytochrome P-450 1A1/1A2. These cytochrome forms were detected in low concentrations in hepatocytes, macrophages of the small intestinal villi and mesenteric and mediastinal lymph nodes of intact animals. Basal induction of monooxygenase enzymes can be caused by cytochrome inductors entering with air (environmental pollution) or food.

The Possibility of Cytochrome P450 1A1/1A2 Induction in Cells of Distant Lymph Nodes of Rats after Enteral Treatment with Benzo[a]pyrene

The mesenteric, mediastinal, and popliteal lymph nodes of rats were studied by indirect immunoperoxidase method using monoclonal antibodies to cytochrome P450 1A1/1A2 after oral treatment with benzo[a]pyrene. These cytochrome forms were detected in monocytes, macrophages, reticular and littoral cells, cell detritus, and liquid contents of the paracortical and medullary sinuses of all studied lymph nodes. The results indicate that exo- and endogenous toxic substances are oxidized not only in the liver, but also in lymph nodes.

Limb Lymph Node Response to Bone Fracture

In previous clinical studies, dilation of afferent lymphatics and enlargement of inguinal lymph nodes (LN) were observed in lymphoscintigrams from patients with persistent posttraumatic edema of lower extremities after fractures and trauma of soft tissues. In this study, changes in rat popliteal and iliac lymph nodes draining lymph from the site of tibial fracture and adjacent soft tissue injury were investigated. The observed parameters were lymph node weight, cell number, phenotype frequency, cell cytokine expression, and reactivity to mitogens. The key observations included: a) increase in the weight and total cell number of the lymph nodes; b) increased autotransformation rate and responsiveness of lymph node cells to mitogen; c) decreased frequency of ED1 macrophages and activated OX8 cytotoxic cells in flow cytometry analysis; d) high expression of OX6 class II-positive, OX7 (stem cells), OX62 (migrating dendritic cells), ED1 (macrophages), and OX12 (B cells) on immunohistochemical sections of LNs with some few HIS48 (granulocytes); e) high expression of NOS3 and TGF beta by lymph node lymphocytes and endothelial cells. In summary, local lymph nodes reacted to internal wounds, such as bone fracture and injury to adjacent tissues, through mobilization of cells from the blood circulation, along with activation of cellular subsets. The molecular mechanism that provides the signal for this reaction remains unknown. The absence of major changes in the frequency of lymph node cell subpopulations indicates that lymph nodes are constitutively prepared for influx of antigens from damaged tissues and react only with increase in cell number and cell activation. The nature of the reaction, including lack of immunization against autoantigens, remains unclear. Further elucidation will require studies on the mechanism of cross-tolerance to self-antigens during wound healing.

A protective effect of early pregnancy factor on experimental autoimmune encephalomyelitis induced in Lewis rats by inoculation with myelin basic protein

Experimental autoimmune encephalomyelitis (EAE) is an organ-specific autoimmune disease characterised by inflammation and demyelination of the central nervous system and is the best available animal model of multiple sclerosis (MS). Since previous studies have shown that EAE is less severe or is delayed in onset during pregnancy and that administration of the pregnancy hormone early pregnancy factor (EPF) down-regulates EAE, experiments in the present study were designed to explore further the role of EPF in EAE. By using the rosette inhibition test, the standard bioassay for EPF and, by semi-quantitative RT-PCR techniques, we have now shown that inflammatory cells from the spinal cord of rats with EAE can produce and secrete EPF, with production being greatest during recovery from disease. Administration of EPF to rats with EAE resulted in a significant increase in the expression of IL-4 and IL-10 mRNA and a significant decrease in IFN-γ mRNA expression in spinal cord inflammatory cells. Encephalitogenic MBP-specific T cell lines were prepared from popliteal lymph nodes of rats with EAE. Proliferation assays using these cells demonstrated the ability of exogenous EPF to down-regulate the responses of T lymphocytes to MBP.

Changes in lymphokine receptor expression and fatty acid composition of phospholipids and triacylglycerols in rat adipocytes associated with lymph nodes following a transient immune challenge.

Single-photon counting fluorimetry was used to record the time course of the expression of interleukin-10 receptors labelled with fluorescent antibodies on the surface of adipocytes over 24h, following an immune challenge to the rat popliteal lymph node. Homologous perinodal and remote-from-node samples from the stimulated and unstimulated popliteal depots were compared in rats fed on plain chow and chow supplemented with 10% w/w suet, fish or vegetable oils. Receptor expression was maximal 6 h after stimulation, and returned to baseline after 24 h, and was similar in the stimulated and unstimulated depots. Fewer receptors were elicited in tissues from rats fed lipid-supplemented diets compared with the control diet, with fewest of all following the fish oil diet. These data suggest that interleukin-10 is involved in local interactions between perinodal adipocytes and lymph node lymphoid cells. Both triacylglycerols and phospholipids contained more polyunsaturates and fewer saturates in perinodal adipose tissue than in samples from sites not associated with lymphoid tissue. These data are consistent with paracrine interactions between perinodal adipocytes and activated lymphoid cells.

Immune potential of lymph node-derived lymphocytes in uncontrolled hemorrhagic shock.

The state of T cell immunity was evaluated in rats in early (1-4 h) hemorrhagic shock induced by a massive splenic injury. T cell subpopulations from treated and untreated shocked animals were tested by flow cytometry and the results were compared with healthy controls. A fall in CD4+ T lymphocyte and natural killer (NKR-P1+) cell number, marked decline in the T helper (CD4+) to T suppressor (CD8+) ratio, and decrease of interleukin-2 receptor (IL-2R) bearing cells in peripheral blood, mesenteric, and popliteal lymph nodes of rats was found in the early stages of hemorrhagic shock. The same phenotype profile was also revealed in lymphocytes of rats in hemorrhagic shock following massive splenic injury treated with Ringer's lactate. The number of TCRalpha beta and TCR-gamma delta positive cells, as well as the percentage of CD4 and CD8 positive cells in the thymus, was similar in all groups of rats. Culture of lymph node cells taken from rats following hemorrhage in the presence of 100 U/mL hrIL-2 resulted in a marked increase in the number of NKR-PI+ positive cells from 4.2% to 30.5% (P < 0.001). Magnet separated NKR-P1+ fractions lysed the allogeneic fibroblasts in the same manner as IL-2-activated NKR-P1 cells from the control rats. Popliteal lymph node (PLNi) CD8b+ lymphocytes from rats in hemorrhagic shock preinfected into the footpad with cytomegalovirus (CMV) 6 days prior to injury lost their ability to lyse the CMV-infected fibroblasts and protect the monolayer from CMV infection when compared with PLNi cells from control infected rats. The possible mechanisms for the observed cellular dysfunction following hemorrhage are discussed.

Discovery of Ascomycin Analogs with Potent Topical but Weak Systemic Activity for Treatment of Inflammatory Skin Diseases

Drug therapy for the major inflammatory skin diseases, which include atopic dermatitis, psoriasis and allergic contact dermatitis, is often inadequate due to poor efficacy, toxicity, or both. Much research has focused on the macrolactam T cell inhibitors as a promising new class of agents for immunotherapy, and medicinal chemistry efforts to design novel ascomycin analogs have produced clinically promising agents. A synthetic program to modify the ascomycin nucleus to alter its physicochemical properties and promote systemic clearance is described. A biologic screening strategy to identify analogs with reduced systemic activity and rapid pharmacokinetic elimination led to identification of the clinical candidate, ABT-281. A swine contact hypersensitivity model was used as a stringent indicator of skin penetration as human doses of topical corticosteroids produced inhibition only in the 50% range and ED50 values were 100-fold less potent than in rat. Also, cyclosporine was confirmed to be topically inactive in swine, as seen in human. ABT-281 had topical potency equal to tacrolimus (FK506) despite a severalfold lower potency for inhibiting swine T cells in vitro, consistent with superior skin penetration. ABT-281 was found to have a shorter duration of action after i.v. dosing in monkeys using an ex vivo whole blood IL-2 production assay. Systemic potency was reduced by 30-fold or more in rat popliteal lymph node hyperplasia and contact hypersensitivity assays. Following i.v. or i.p. administration in the swine contact hypersensitivity model, ABT-281 was 19- and 61-fold less potent, respectively, than FK506. Pharmacokinetic studies showed that ABT-281 had a shorter half life and higher rate of clearance than FK506 in all three species. The potent topical activity and reduced systemic exposure of ABT-281 may thus provide both efficacy and a greater margin of safety for topical therapy of skin diseases.

Studies on an immunosuppressive macrolactam, ascomycin: synthesis of a C-33 hydroxyl derivative.

Ascomycin 2, a close analogue of the immunosuppressant FK506 1, was modified to incorporate a hydroxyl group at the C-33 position. This increased the aqueous solubility of ascomycin by a hundred-fold at pH 7.4 and by approximately 300-fold at pH 6.5. Ascomycin 3 also exhibited an excellent immunosuppressive activity in vitro, as tested in a human mixed lymphocyte proliferation (HuMLR) assay, and in vivo using a rat popliteal lymph node (rPLN) hyperplasia assay.

The distribution, immune complex trapping ability and morphology of follicular dendritic cells in popliteal lymph nodes of aged rats.

Immune system function declines with age, and lymph nodes involute. The aims of the study were to describe the distribution of follicular dendritic cells (FDCs) in the lymphoid follicles of aged rats, and to determine whether these cells have reduced ability to trap immune complexes (ICs). Popliteal lymph nodes of rats aged 24-28 months were immunostained for S-100 protein as a marker of FDCs. Some rats were pretreated with peroxidase-anti-peroxidase complex (PAP) as an IC. FDCs were densely distributed in lymphoid follicles, which contained many primary follicles and a few secondary follicles. FDCs in primary follicles stained weakly for S-100 protein, and showed weak trapping, while those in secondary follicles stained strongly for both S-100 protein and trapping. Ultrastructurally, in involuted lymphoid follicles FDCs were atrophic. We conclude that FDCs in aged rats are densely distributed in involuted follicles and show reduced trapping ability and atrophic morphology. This seems to reflect the long life of FDCs and the reduced numbers of lymphoid cells in these follicles. We suggest that FDCs in aged rats may show some of their normal functions if fully developed germinal centers are induced, and may not play an important role in the process of involution of the follicles.

T Cell Reactivity to Acetylcholine Receptor in Rats Orally Tolerized against Experimental Autoimmune Myasthenia Gravis

Myasthenia gravis and its animal model experimental autoimmune myasthenia gravis (EAMG) represent T cell-dependent autoimmune diseases mediated by antibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction. Oral administration of Torpedo AChR to Lewis rats prior to immunization with the myasthenogenic Torpedo AChR and complete Freund's adjuvant results in the prevention of clinical EAMG, and the suppression of AChR-specific B cell responses. To examine the influence of oral tolerance to EAMG on AChR-reactive T cells, we determined and enumerated such cells in the popliteal, inguinal, and mesenteric lymph nodes, spleen, and thymus by a T cell immunospot assay that is based on the secretion of interferon-γ (IFN-γ) by antigen-reactive T cells. A diminution of such cells was detected in popliteal, inguinal, and mesenteric lymph nodes of rats orally tolerized to EAMG compared to unfed or vehicle-fed animals. We conclude that oral administration of AChR, in addition to preventing clinical signs of EAMG and suppressing AChR-specific B cell responses, also counteracts the development of AChR-reactive INF-γ-secreting cells in certain lymphoid organs.

In vivo inhibition of the rat primary antibody response to antigenic stimulation by somatostatin.

Somatostatin inhibits in vitro lymphocyte proliferative responses from a variety of species including human, mouse and rat. The immunoinhibitory effects of somatostatin are thought to involve binding to specific cell surface somatostatin receptors on immunocompetent cells. This report describes an in vivo immunoinhibitory effect of somatostatin on the rat popliteal lymph node lymphocyte primary antibody response to sheep red blood cell (SRBC) stimulation. Infusion of somatostatin immediately following SRBC injection into the hind feet of rats had a dose-related inhibitory effect. At the highest concentration used, 10 mumol/L, the level of inhibition was similar to that previously described following neonatal capsaicin treatment of rats. This suggests that neonatal capsaicin treatment may lead to decreased primary antibody responses to SRBC by a selective effect on tachykinin containing nerves and a lesser effect on somatostatin containing nerves. The immunoinhibitory effect of somatostatin was reversed by co-infusion of neurokinin A but not substance P, both of which have been shown to stimulate this response. This suggests the possibility that multiple tachykinin receptors are involved in the modulation of the SRBC primary antibody response in vivo. These results present evidence for an in vivo immunomodulatory role of somatostatin.

The enveloping of intercellular collagenous fibrils by reticular cell processes in postnatal development of rat lymph nodes.

In the lymph nodes of adult rats reticular fibers are known to be covered by the processes of reticular cells. This study aims to visualize the sequence of the envelopment of reticular fibers by reticular cells during development. Rat popliteal lymph nodes of one to twenty-three days after birth were examined by electron microscopy. At the earliest stages, collagenous protofibrils were found in the intercellular space between studded mesenchymal cells. The protofibrils clustered around the plasma membrane of immature reticular cells and then became arranged into microfibrils of 30-40 nm in diameter. Bundles of the fibrils which might be called reticular fibers were surrounded by processes of more than one reticular cell. Then the reticular fiber came to be enclosed by the cytoplasmic process of a single reticular cell. Finally at 16-23 days after birth, the reticular fiber was completely ensheathed by the thick cytoplasmic process of a single reticular cell closed with a junctional complex. Throughout these periods, basal lamina-like materials existed between the reticular fiber and cytoplasmic process. Clumps of fibrils were rarely in contact with leukocytes, including lymphocytes. Immature elastic fibers appeared among collagenous fibrils of the reticular fiber when the fiber came to be enclosed by processes of some reticular cells. It was shown that the enclosure of the reticular fiber by the reticular cell did not result from physical pressure due to the increase of the number of lymphocytes, but the reticular cell actively enclosed the reticular fiber.(ABSTRACT TRUNCATED AT 250 WORDS)

Macrophages and the activity of high endothelial venules. The effect of interferon‐γ

Interruption of the afferent lymphatic vessels of rat popliteal lymph nodes led to the disappearance of the monoclonal antibody ED3-reactive subcapsular sinus macrophages within 3 weeks, but had no effect on the ED1+ macrophages in the paracortical area. This disconnection of the afferent lymph flow to the popliteal lymph node also reduced the capacity of high endothelial venules (HEV) to bind lymphocytes and led to a flattening of the HEV. Activating factors or cells in the incoming lymph might be responsible for the maintenance and function of several different cell populations and we therefore wished to determine if the effects of interruption could be restored by injection of recombinant rat interferon-gamma (rIFN-gamma). Injection of rIFN-gamma directly into operated lymph nodes could mediate an apparent increase of ED1+ cells within 24 h but rIFN-gamma could not restore the macrophage subpopulation in the subcapsular sinus, as recognized by monoclonal antibody ED3. Restoration of the decreased binding capacity of HEV could not be observed with the doses and time points tested, suggesting that HEV are a distinct type of endothelium.

リンパ濾胞内抗原捕捉域の変遷に関する三次元的研究

In lymph follicles of rat popliteal lymph nodes (PLNs), the transition of the localization of PAP (peroxidase-anti-peroxidase complex) trapping area with development of germinal centers was tridimensionally studied to analyze the differentiation of follicular dendritic cells (FDCs). Rats 5 weeks old were injected sheep red blood cells (SRBCs) into foot pads. The PLNs were extirpated at different intervals, at one day before extirpations PAP were injected into foot pads. Detection of PAP in lymph follicles and detail observations were performed in serial paraffin sections and in electron microscopy by immunoperoxidase technique. After the injection of SRBCs, lymph follicles and germinal centers increased in number. In early primary lymph follicles, the PAP trapping area occupied large space, however the ratio of the PAP trapping area occupying in the follicles was decreased with development of germinal centers. The trapping of PAP became weak in which centroblasts had been just appeared in the primary follicles, and then the medullary side of the germinal centers revealed no PAP trapping with further development of germinal centers. In electron microscopical observation, the trapping of PAP in secondary follicles could be found on the surface of cytoplasmic membranes of FDSs, which formed complicated labyrinth structures, in corona and light zone. On long time interval after injection of SRBCs, the germinal centers were atrophic, while cytoplasmic processes of FDCs remained complicated. The differentiation of FDCs is discussed.

The early postnatal development of the primary immune response in rat popliteal lymph node, stimulated with thymus-independent type-1 and type-2 antigens

To examine the development of the postnatal immune response to thymus-independent type-1 (TI-type 1) and TI type-2 antigens, respectively, trinitrophenyl-lipopolysaccharide (TNP-LPS) or TNP-Ficoll was injected subcutaneously into the hind footpads of young rats of various ages. After 5 days the popliteal lymph nodes (PLNs) were removed and the localization pattern of specific anti-TNP antibody-containing cells was studied. The first specific antibody-containing cells elicited in rats by TNP-LPS appeared in animals at day 19 after birth. The results suggest that the development of these cells from lymphocyte to plasma cell occurs while they migrate from cortex to medulla. An unexpected finding was the low response to TNP-Ficoll in PLN; from 6 weeks after birth only very few specific antibody-containing cells were found. However, in the spleen numerous anti-TNP antibody-containing cells were found in the periarteriolar lymphocyte sheaths. To test the exclusive role of the spleen in the appearance of anti-TNP antibody-containing cells in lymph node after subcutaneous administration of TNP-Ficoll, the experiment was repeated in rats that had been splenectomized. Evidence from these experiments suggests that the spleen plays a major role in the appearance of the above-mentioned cells in lymph nodes.

THE ENTRY OF LYMPHOCYTES INTO STIMULATED LYMPH NODES THE SITE OF SELECTION OF ALLOANTIGEN-SPECIFIC CELLS

During a primary response to antigen, lymph nodes (LN) receive antigen-specific lymphocytes (ASL) from the recirculatory pool via specialized postcapillary venules (SPCV). Preliminary experiments demonstrated a three-fold increase in entry of 51Cr-labeled thoracic duct lymphocytes (TDL) into the rat popliteal LN 3-4 days after alloantigenic stimulation. This increased entry was accounted for by an associated 4-5-fold increase in LN blood flow. It was unclear whether recruitment of ASL occurred within the SPCV lumen leading to selective entry of ASL--or, more conventionally, that entry of lymphocytes was a random process and that recruitment of ASL occurred in the extravascular space by selective retention. Thus a double-labeling technique was used to study the migratory characteristics of two populations of thoracic duct lymphocytes (TDL) that had been passaged from blood to lymph. The first population, passaged through a semiallogeneic animal, was negatively selected, and thus 99% depleted of ASL; the second population, passaged through a syngeneic animal, contained a normal complement of naive ASL. The two populations were combined before i.v. injection into syngeneic recipients. At 24 hr the right popliteal LN, which had previously been stimulated with the specific antigen, showed a preferential localization of the TDL containing ASL compared with the left (unstimulated) popliteal LN. This preferential localization was not present at 1 hr after injection. In the light of background data on the kinetics of lymphocyte traffic it is concluded that ASL were selected not at the luminal surface of SPCV but in the LN after they had left the bloodstream.

The host component of the popliteal lymph node graft-versus-host reaction. Selective representation of lymphocyte subsets and the requirement for alloantigenic incompatibility between donor cells and activated host B cells.

We have examined the cellular changes taking place in rat popliteal lymph nodes undergoing a graft-versus-host (GvH) reaction. Examination of immunoperoxidase-stained lymph node sections, using a panel of mouse monoclonal antibodies directed against different rat lymphoid cell subsets, revealed a disorganization of the lymph node architecture with disappearance of the follicles, and an intermingling of T and B cells, so that no distinct T- and B-cell areas were visible any more. Since the GvH nodes showed a preferential accumulation of host B cells over host T cells (particularly over the W 3/25+ T helper cell subset), we also investigated the requirements for host B cell activation. The popliteal lymph node GvH reaction was induced in (PVG X DA)F1 rats by the injection of PVG cells into one foot and by DA cells into the other foot, and then immunoglobulin kappa allotype marked PVG B cells from athymic donors were injected intravenously. The allotype marked B cells proliferated vigorously in response to the DA T cells, but much less in response to the PVG T cells. These results indicate that the massive B-cell activation taking place in GvH reactions may require an alloantigen incompatibility between donor T cells and host B cells, and argue against non-specific mitogenic induction of the B cells.