Concurrent production of interleukin-2, interleukin-10, and gamma interferon in the regional lymph nodes of mice with influenza pneumonia.

Cytokine production has been assessed at the single-cell level (ELISPOT assay) for freshly isolated mediastinal lymph node cells from C57BL/6 mice with primary, nonfatal influenza pneumonia. The mediastinal lymph node populations were also secondarily stimulated in vitro, and culture supernatants were assayed by enzyme-linked immunosorbent assay. Both approaches showed minimal evidence of protein secretion for interleukin-4 (IL-4), IL-5, and tumor necrosis factor, while IL-2, IL-10, and gamma interferon (IFN-gamma) were prominent throughout the response. The numbers of IL-2- and IFN-gamma-producing cells were maximal at 7 days after infection, while the total counts for cells secreting IL-10 were fairly constant from day 3 to 7. The cultures that were stimulated with virus in vitro showed in inverse relationship between IL-10 and IFN-gamma production, with IL-10 peaking on day 3 and IFN-gamma peaking on day 7. Lymphocytes secreting IL-2, IL-10, and/or IFN-gamma were present in CD4+ and CD8+ populations separated by fluorescence-activated cell sorting, although the CD8+ T cells produced less cytokine and were at a relatively lower frequency. Addition of recombinant IL-10 to the virus-stimulated cultures decreased the amount of IFN-gamma that could be detected, while incorporation of a monoclonal antibody to IL-10 had the opposite effect. A neutralization experiment also indicated that IL-2 was the principal mediator of lymphocyte proliferation. These experiments thus show that the developing T-cell response in the regional lymph nodes of mice with influenza cannot be rigidly categorized on the basis of a TH1 or TH2 phenotype and suggest possible regulatory mechanisms.     

The kinetics and stimulant dependence of cytokine production by blood and bronchoalveolar lavage T cells evaluated at the single cell level.

We have previously shown that bronchoalveolar lavage (BAL) T cells from human airways predominantly produce interferon gamma (IFN-gamma) and interleukin 2 (IL-2) when stimulated ex vivo. The kinetics of TH1 and TH2 cell cytokine production by T cells from both blood and BAL were studied to establish the optimal time after stimulation either with pharbol myristate (PMA) and ionomycin or with the more physiological stimulus of anti-CD3 for intracellular cytokine detection of IFN-gamma, IL-2, IL-4 and IL-5 in both blood and BAL T cells. The optimal time for positive identification of IL-2 in both blood and BAL was 5 h after PMA/ionomycin stimulation, whereas the first peak for IFN-gamma was found after 5 h in blood but after only 3 h in BAL. T cells from different biological compartments responded differently to each of the stimuli. Whilst anti-CD3 stimulation did not induce TH1 cytokine production in blood T cells, it readily induced both IFN-gamma and IL-2 production in BAL T cells. The kinetics of cytokine production were found to be stimulus dependent. Whilst IL-2 production showed similar kinetics with both stimuli, the kinetics of IFN-gamma production differed between stimuli. We have also examined the effect of five different stimuli on cytokine production by T cells to determine whether different forms of stimulation may selectively stimulate or inhibit different cytokines. Not surprisingly, PMA/ionomycin induced a greater percentage of BAL T cells to produce TH1 cytokines. However, other than modest amounts of the TH2 cytokines IL-4 and IL-5 were not induced by any of the five stimuli.     

Different cytokine patterns correlate with the extension of disease in pulmonary tuberculosis.

The relative amounts of different pro- and anti-inflammatory cytokines released at the site of infection by bronchoalveolar lavage (BAL) cells may influence the presentation of tuberculosis. To investigate this hypothesis the in situ release by BAL cells of the following cytokines was measured and correlated with the chest X-ray findings of 43 patients with pulmonary tuberculosis: interleukin (IL)-8, macrophage inflammatory protein-1alpha (MIP-1alpha), IL-6, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta), interferon-gamma (IFN-gamma), IL-2, IL-4 and IL-5. The release of IL-8 and IL-6 decreased with the progression of the disease, while the release of MIP-1alpha was increased in patients with advanced tuberculosis. The release of TNF-alpha and TGF-beta did not differ between patients with or without cavitary lesions. The Th1 (IFN-gamma and IL-2) and Th2 (IL-4 and IL-5) cytokine release exhibited a gradual increment with the advance of tuberculosis. Thus, our data provide evidence that a Th0 cytokine pattern is predominant at the site of pulmonary tuberculosis. In conclusion, immunoparalysis status could not be observed in our patients with severe tuberculosis.     

Polarized type 1 cytokine profile in bronchoalveolar lavage T cells of patients with hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is characterized by an inflammatory lymphocytic alveolitis comprised of both CD8+ and CD4+ T cells. Animal models suggest that HP is facilitated by overproduction of IFN-gamma, and that IL-10 ameliorates severity of the disease, indicating a Th1-type response. To determine whether a Th1 phenotype in HP also exists clinically, bronchoalveolar lavage (BAL) and peripheral blood (PB) T cells were obtained from HP individuals and analyzed for Th1 vs Th2 cytokine profiles. It was determined that soluble OKT3-stimulated BAL T cells cocultured with alveolar macrophages produced more IFN-gamma and less IL-10 than PB T cells cocultured with monocytes, but no difference was observed in IL-4 production. The monocytic cells did not account for this difference, as CD80 and CD86 expressions were similar, and coculturing PB T cells with alveolar macrophages resulted in no difference in IFN-gamma production. Similarly, there was no difference in IL-12 production between stimulated BAL or PB T cells; however, addition of rIL-12 significantly increased production of IFN-gamma by BAL T cells, but not by PB T cells. This effect was due to a difference in IL-12R expression. High affinity IL-12R were only present in association with BAL T cells. These studies indicate that clinical HP is characterized by a predominance of IFN-gamma-producing T cells, perhaps resulting from a reduction in IL-10 production and an increase in high affinity IL-12R compared with blood T cells.     

Immunological determinants of ventilatory changes induced in mice by dermal sensitization and respiratory challenge with toluene diisocyanate

The objective of the study was to characterize better the immunologic mechanisms underlying a previously developed animal model of chemical-induced asthma. BALB/c and severe combined immunodeficiency disease (SCID) mice received toluene diisocyanate (TDI) or vehicle on each ear on day 1 and/or day 7. On day 10, they were intranasally challenged with TDI or vehicle. Ventilatory function was monitored by whole body plethysmography for 40 min after challenge. Reactivity to methacholine was measured 23 h later: enhanced pause and actual resistance measurements. Pulmonary inflammation was assessed 1, 6, and 24 h after challenge by bronchoalveolar lavage (BAL). Tumor necrosis factor-alpha and macrophage inflammatory protein (MIP)-2 levels were measured in BAL. Immunological parameters included total IgE, IgG1, and IgG2a in serum, lymphocyte populations in auricular and cervical lymph nodes, and IL-4 and IFN-gamma levels in supernatants of lymph node cells, cultured with or without concanavalin A. Ventilatory changes suggestive of airway obstruction and increased methacholine reactivity were observed in all TDI-sensitized and TDI intranasally instilled mice, except in SCID mice. A neutrophil influx, accompanied by an increase in MIP-2 levels, was found in BAL of all responding groups 6 and 24 h after intranasal challenge. In BALB/c mice an increased level of CD19+ B cells was found in the auricular lymph nodes. IL-4 and IFN-gamma levels were increased in supernatants of concanavalin A-stimulated auricular lymph node cells from BALB/c mice completely treated with TDI. These results indicate that our model is dependent on the presence of lymphocytes, but it is not characterized by a preferential stimulation of Th1 or Th2 lymphocytes.     

Murine cytomegalovirus infection alters Th1/Th2 cytokine expression, decreases airway eosinophilia, and enhances mucus production in allergic airway disease

Concomitant infection of murine CMV (MCMV), an opportunistic respiratory pathogen, altered Th1/Th2 cytokine expression, decreased bronchoalveolar lavage (BAL) fluid eosinophilia, and increased mucus production in a murine model of OVA-induced allergic airway disease. Although no change in the total number of leukocytes infiltrating the lung was observed between challenged and MCMV/challenged mice, the cellular profile differed dramatically. After 10 days of OVA-aerosol challenge, eosinophils comprised 64% of the total leukocyte population in BAL fluid from challenged mice compared with 11% in MCMV/challenged mice. Lymphocytes increased from 11% in challenged mice to 30% in MCMV/challenged mice, and this increase corresponded with an increase in the ratio of CD8(+) to CD4(+)TCRalphabeta lymphocytes. The decline in BAL fluid eosinophilia was associated with a change in local Th1/Th2 cytokine profiles. Enhanced levels of IL-4, IL-5, IL-10, and IL-13 were detected in lung tissue from challenged mice by RNase protection assays. In contrast, MCMV/challenged mice transiently expressed elevated levels of IFN-gamma and IL-10 mRNAs, as well as decreased levels of IL-4, IL-5, and IL-13 mRNAs. Elevated levels of IFN-gamma and reduced levels of IL-5 were also demonstrated in BAL fluid from MCMV/challenged mice. Histological evaluation of lung sections revealed extensive mucus plugging and epithelial cell hypertrophy/hyperplasia only in MCMV/challenged mice. Interestingly, the development of airway hyperresponsiveness was observed in challenged mice, not MCMV/challenged mice. Thus, MCMV infection can modulate allergic airway inflammation, and these findings suggest that enhanced mucus production may occur independently of BAL fluid eosinophilia.     

Influences of orally administered lactoferrin on IFN-gamma and IL-10 production by intestinal intraepithelial lymphocytes and mesenteric lymph-node cells.

Intestinal mucosal immunity plays an important role in mucosal and systemic immune responses. We investigated the influences of orally administered bovine lactoferrin (LF) on cytokine production by intestinal intraepithelial lymphocytes (IEL) and mesenteric lymph-node (MLN) cells, especially T cells. Bovine LF or bovine serum albumin (control) was administered to mice once daily for 3 d. After 24 h from the last administration, IEL of the jejunum and ileum and MLN cells were isolated. These cells were cultured with and without the anti-T-cell-receptor antibody, and then the culture supernatants were assayed for cytokines with ELISA. Oral LF did not affect the ratio of T-cell subpopulations in IEL and MLN; however, LF enhanced both interferon (IFN)-gamma and interleukin (IL)-10 production by unstimulated IEL and by IEL stimulated with the alphabeta T-cell receptor but not with the gammadelta T-cell receptor. LF also enhanced both IFN-gamma and IL-10 production by stimulated and unstimulated MLN cells. The production level of IFN-gamma by MLN cells was correlated with that of IL-10. These results suggest that oral LF enhances the production of both Th1-type and Th2/Tr-type cytokines in the small intestine of healthy animals.     

Endotoxin and Staphylococcus aureus enterotoxin A induce different patterns of cytokines.

The effects of Staphylococcus aureus enterotoxin A (SEA) and lipopolysaccharide (LPS) in cytokine production were assessed at the single cell level in cells obtained from healthy blood donors. Cytokine production was studied with UV-microscopy of fixed and permeabilized cells stained with cytokine specific monoclonal antibodies. The cytokines evaluated included tumour necrosis factor (TNF)-alpha, interleukin (IL)-1 alpha, IL-1 beta, IL-6, IL-8, IL-10, IL-2, IL-4, interferon (IFN)-gamma and TNF-beta. LPS exhibited marked production of IL-1 alpha, IL-1 beta, TNF-alpha, IL-6 and IL-8. After LPS stimulation IL-1 alpha, IL-1 beta, TNF-alpha and IL-8 were the dominating products, all peaking at or before 4 hours after cell stimulation. In addition, IL-10 production was evident after 12 hours of cell stimulation. The T-lymphocyte-derived cytokines TNF-beta, IL-2, IFN-gamma and IL-4 were never detected in the cultures. All cytokine production, except IL-8, was downregulated at 96 hours. In contrast, peak production of IL-1 alpha, IL-1 beta and IL-8, which were the dominant products, occurred after 12 hours in the SEA-stimulated cultures. Further, a significant T-lymphocyte production of TNF-beta, TNF-alpha, IFN-gamma and IL-2 was found with peak production 12-48 hours after initiation. Only low amounts of IL-6 were evident. The two types of cytokine pattern and kinetics found may correspond to the different clinical conditions after invasive Gram-negative Escherichia coli vs Gram-positive Staphylococcus aureus infections in humans, with a much more rapid onset of disease after E. coli infections.(ABSTRACT TRUNCATED AT 250 WORDS)     

Increased specific T cell cytokine responses in patients with active pulmonary tuberculosis from Central Africa

An understanding of T cell responses that are crucial for control of Mycobacterium tuberculosis (MTB) has major implications for the development of immune-based interventions. We studied the frequency of purified protein derivative (PPD)-specific CD3) cells expressing interleukin-2 (IL)-2, gamma interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and IL-10 in HIV-negative pulmonary tuberculosis patients (TB, n=30) as well as in healthy individuals (controls, n=21) from Central Africa. Increased frequencies of PPD-stimulated CD3+ cells expressing IL-2, IFN-gamma, and TNF-alpha in TB were seen when compared with frequencies of controls. The presence of type 1 cytokine biased responses in TB patients was supported by a shift in the distribution pattern of cytokine expression from exclusively IL-2 or TNF-alpha expression seen in controls towards an increased frequency of IFN-gamma/IL-2 or IFN-gamma/TNF-alpha co-expression in TB. Higher levels of PPD-induced IFN-gamma in the supernatants from TB patients than from controls were found, which correlated with its intracellular expression. PPD was a weak inducer of IL-10 in T cells and insufficient in promoting cytokine production in TCRgammadelta+CD3+ cells. Non-specific stimulation with PMA and ionomycin revealed increased frequencies of CD4+ cells expressing IFN-gamma in controls, while expression of IL-2, IL-4, IL-10, IL-13, and TNF-alpha was not different. Non-specific cytokine responses of TCRgammadelta+CD3+ cells were similar in all groups. Pulmonary TB in Central Africa is associated with enhanced expression and secretion of specifically induced cytokines that are frequently implicated in host defense against MTB.     

Cytokine detection by multiplex technology useful for assessing antigen specific cytokine profiles and kinetics in whole blood cultured up to seven days

Cytokine profile assessment is important to characterize immune responses to pathogens. To identify optimal time points for determination of cytokine profiles, we diluted whole blood 1:10, to enable daily cytokine measurements during one week. Cultures for 10 blood donors were set up in the presence of phytohemagglutinin (PHA), cytomegalovirus (CMV) or Candida. Supernatant levels of interleukin-2 (IL-2), IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-17, interferon-gamma (IFN-gamma), granulocyte/macrophage colony-stimulating factor, and tumor necrosis factor-alpha (TNF-alpha), were determined by multiplex technique, and intracellular cytokine staining (ICS) was employed to detect IFN-gamma, IL-2, IL-4 and IL-13 in CD3+ cells. The multiplex analysis detected representative cytokine profiles for the majority of the cytokines on day 7 by identifying peak levels or good correlation with peak levels, with the exception of IL-2 and TNF-alpha in PHA and CMV cultures and IL-10 in PHA cultures. For these cytokines an extracellular measurement on day 2-3 would be appropriate. The intracellular cytokines showed distinct kinetics for IFN-gamma and IL-2, while IL-4 and IL-13 were not detected at all with ICS. In conclusion, the combination of whole blood cultures with multiplex analysis is a simple and powerful tool that can be used to identify detailed cytokine profiles of specific cell-mediated immune responses.     

Orally administered Kampo (Japanese herbal) medicine, "Juzen-Taiho-To" modulates cytokine secretion in gut associated lymphoreticular tissues in mice.

The influence of the oral administration of Juzen-Taiho-To (JTT; Si-Quan-Da-Bu-Tang in Chinese), a Kampo (Japanese herbal) medicine, on the cytokine production in mice were investigated. Lymphocytes from spleen (SPN), mesenteric lymph node (MLN) and Peyer's patches (PP) from mice, which received orally administered JTT for 2 weeks, were stimulated with concanavalin A (Con A), and the resulting conditioned medium was tested for cytokine production by ELISA. Administration of JTT caused enhancement of interferon g (IFN-gamma) and interleukin-4 (IL-4) production to some extent but decreased IL-5 production from the SPN. On the other hand, notable changes in cytokine production were observed in lymphocytes from MLN and PP. Administration of JTT increased IFN-gamma production remarkably, as well as IL-5 secretion from both MLN and PP, whereas IL-2 secretion was plainly reduced. The ratio of IFN-gamma and IL-4 was shifted to Th1 dominant in MLN and PP, however changed little in SPN. The flow cytometric analysis revealed that the population of CD3+, CD4+, CD45R/B220+, and CD45RBlowCD4+ cells in each lymphocyte was not changed significantly after oral administration of JTT. These findings demonstrate that the lymphocytes from gut associated lymphoreticular tissues (GALT) are more sensitive to produce cytokines on cytokine production than those from SPN by oral administration of JTT, and that the modulation of cytokine production may be due to functional change of lymphocytes but not change in lymphocytes population.     

Expression of the Th1 chemokine IFN-gamma-inducible protein 10 in the airway alters mucosal allergic sensitization in mice

Although the preliminary characterization of chemokines and their receptors has been prolific, comparatively little is known about the role of chemokines in the evolution of immune responses. We speculate that the preferential recruitment of a particular immune cell population has implications for the short- and long-term features of an adaptive response. To test this hypothesis, we employed adenovirus-mediated gene transfer to express the Th1-affiliated, CXC chemokine IFN-gamma-inducible protein (IP) 10 in the airways of mice undergoing a mucosal sensitization regimen known to result in a Th2-polarized allergic response. This resulted in a approximately 60-75% inhibition of eosinophils in the bronchoalveolar lavage (BAL); these inflammatory changes were accompanied by enhanced IFN-gamma, ablated IL-4, and, peculiarly, unaltered IL-5 and eotaxin levels in the BAL. The effect of IP-10 expression was shown to be dependent on IFN-gamma, as there was no statistically significant reduction in BAL eosinophilia in IFN-gamma knockout mice subjected to the IP-10 intervention. Flow cytometric analysis of mononuclear cells in the lung revealed a approximately 60% reduction in the fraction of CD4(+) cells expressing T1/ST2, a putative Th2 marker, and a parallel increase in the proportion expressing intracellular IFN-gamma following IP-10 treatment. The effect of IP-10 expression at the time of initial Ag encounter is persistent, as mice rechallenged with OVA following the resolution of acute inflammation exhibited reduced eosinophilia and IL-4 in the BAL. Collectively, these data illustrate that local expression of the chemokine IP-10 can introduce Th1 phenomena to a Th2-predisposed context and subvert the development of a Th2 response.     

IL-12 and IL-18 are increased and stimulate IFN-gamma production in sarcoid lungs

Sarcoidosis is a systemic chronic granulomatous disease of unknown cause. Recent investigations revealed that the cytokine profile in inflamed lesions of sarcoidosis is Th1 dominant. To obtain better immunopathologic understanding of sarcoidosis, we examined the expression of IL-12 and IL-18 and their roles in IFN-gamma production in pulmonary sarcoidosis. Sarcoid cases had significantly elevated levels of IL-12 (p40 and p70) and IL-18 in bronchoalveolar lavage (BAL) fluids compared with healthy subjects. IL-12 p70 and IL-18 were immunohistochemically expressed in the epithelioid cells and giant cells of sarcoid granulomas. Significant induction of IFN-gamma, IL-12 p70, and IL-18 was observed from sarcoid BAL fluid cells with LPS stimulation, whereas LPS tended to induce only IL-12 p70 in BAL fluid cells from healthy subjects. Sarcoid cases had significantly greater IFN-gamma induction with LPS stimulation than healthy subjects did. IL-18 mRNA expression was observed in freshly isolated sarcoid BAL fluid cells as well as in LPS-stimulated sarcoid BAL fluid cells, but IFN-gamma and IL-12 mRNA expression was observed only in LPS-stimulated BAL fluid cells. Treatment with anti-IL-12- and anti-IL-18-neutralizing Abs significantly inhibited IFN-gamma production from LPS-stimulated BAL fluid cells of sarcoid cases. Coadministration of rIL-12 or rIL-18 induced greater IFN-gamma production in sarcoid BAL fluid cells than in normal BAL fluid cells. We concluded that bioactive IL-12 and IL-18 were produced in sarcoid BAL fluid cells and synergistically induced IFN-gamma production, indicating important cytokines in the Th1 response of sarcoidosis.     

Early recruitment of neutrophils determines subsequent T1/T2 host responses in a murine model of Legionella pneumophila pneumonia

The contribution of neutrophils to lethal sensitivity and cytokine balance governing T1 and T2 host responses was assessed in a murine model of Legionella pneumophila pneumonia. Neutrophil depletion by administration of granulocyte-specific mAb RB6-8C5 at 1 day before infection rendered mice approximately 100-fold more susceptible to lethal pneumonia induced by L. pneumophila. However, this treatment did not alter early bacterial clearance, despite a substantial decrease in neutrophil influx at this time point. Cytokine profiles in the lungs of control mice demonstrated strong T1 responses, characterized by an increase of IFN-gamma and IL-12. In contrast, neutrophil-depleted mice exhibited significantly lower levels of IFN-gamma and IL-12, and elevation of T2 cytokines, IL-4 and IL-10. Immunohistochemistry of bronchoalveolar lavage cells demonstrated the presence of IL-12 in neutrophils, but not alveolar macrophages. Moreover, IL-12 was detected in lavage cell lysates by ELISA, which was paralleled to neutrophil number. However, intratracheal administration of recombinant murine IL-12 did not restore resistance, whereas reconstitution of IFN-gamma drastically improved bacterial clearance and survival in neutrophil-depleted mice. Taken together, these data demonstrated that neutrophils play crucial roles in primary L. pneumophila infection, not via direct killing but more immunomodulatory effects. Our results suggest that the early recruitment of neutrophils may contribute to T1 polarization in a murine model of L. pneumophila pneumonia.     

Activation pathways implicate anti-HLA-DP and anti-LFA-1 antibodies as lead candidates for intervention in chronic berylliosis

CD4(+) T cells play a key role in granulomatous inflammation in the lung of patients with chronic beryllium disease. The goal of this study was to characterize activation pathways of beryllium-responsive bronchoalveolar lavage (BAL) CD4(+) T cells from chronic beryllium disease patients to identify possible therapeutic interventional strategies. Our results demonstrate that in the presence of APCs, beryllium induced strong proliferation responses of BAL CD4(+) T cells, production of superoptimal concentrations of secreted proinflammatory cytokines, IFN-gamma, TNF-alpha,and IL-2, and up-regulation of numerous T cell surface markers that would promote T-T Ag presentation. Ab blocking experiments revealed that anti-HLA-DP or anti-LFA-1 Ab strongly reduced proliferation responses and cytokine secretion by BAL CD4(+) T cells. In contrast, anti-HLA-DR or anti-OX40 ligand Ab mainly affected beryllium-induced proliferation responses with little impact on cytokines other than IL-2, thus implying that nonproliferating BAL CD4(+) T cells may still contribute to inflammation. Blockade with CTLA4-Ig had a minimal effect on proliferation and cytokine responses, confirming that activation was independent of B7/CD28 costimulation. These results indicate a prominent role for HLA-DP and LFA-1 in BAL CD4(+) T cell activation and further suggest that specific Abs to these molecules could serve as a possible therapy for chronic beryllium disease.     

Distinct cellular immune responses following primary and secondary influenza virus challenge in cotton rats

To evaluate cell-mediated immunity in influenza-infected cotton rats, we compared the cellular composition of spleen, mediastinal lymph nodes (MLN) and bronchoalveolar lavage (BAL) after primary and secondary infection. There was an increase in cellularity in the MLN after primary infection that was further expanded upon rechallenge. CD4(+) T cells expanded after primary infection, but there was preferential increase in the number of CD4-negative T cells following secondary challenge. After primary infection, a large proportion of the monocytes and NK cells were present in the BAL while a T cell population dominated after secondary infection. CD4(+) T cells were predominant in this population unless the animals had been challenged with heterosubtypic influenza A virus. These studies are the first to show evidence of a memory T cell response to influenza infection in cotton rats and show quantitative and qualitative differences between the recall response to homosubtypic and heterosubtypic viruses.     

Early intestinal Th1 inflammation and mucosal T cell recruitment during acute graft-versus-host reaction

Little is understood about the earliest cytokine responses and the role(s) of donor CD4 T cells in the intestine during the induced graft-vs-host reaction (GVHR). We investigated the activation and mucosal homing phenotype of the donor CD4 cells and the kinetics of cytokine responses within the intestine and associated lymphoid tissues during early GVHR. Significant frequencies of donor CD4 cells accumulated within recipient Peyer's patches (PP), mesenteric lymph nodes (MLN), lamina propria (LP), and spleen (SP), during the first 9 days of GVHR. Many donor CD4 cells in SP, MLN, and LP expressed CD44 and also expressed de novo the mucosal homing integrin alpha(4)beta(7) (LPAM-1). A large IFN-gamma response occurred by day 3 in cells from PP and MLN, but much later (day 9) in SP and LP cells. IL-10 production by SP and MLN cells was elevated initially but declined substantially by day 9. IL-4 production by SP, MLN, and PP cells was low on day 3 and showed gradual decline in LP by day 9. IL-5 production by LP cells gradually increased in direct contrast to IL-5 production by MLN cells. The MLN CD4 cells showed the most dynamic changes, with high numbers of activated/effector donor CD4 cells and altered cytokine production consistent with a developing Th1 response. The IFN-gamma responses in PP and MLN preceded that of the SP, suggesting an intestinal origin for some Th1 effector cells in GVHR. Donor CD4 T cells apparently acquire the ability to home to the LP during early GVHR.