Responder Lymph Node Research Articles

Squalene-Based Influenza Vaccine Adjuvants and Their Impact on the Hemagglutinin-Specific B Cell Response.

Influenza infections continue to cause significant annual morbidity and mortality despite ongoing influenza vaccine research. Adjuvants are administered in conjunction with influenza vaccines to enhance the immune response and strengthen protection against disease. Squalene-based emulsion adjuvants including MF59, AS03, and AF03, are registered for administration with influenza vaccines and are widely used in many countries. Squalene-based emulsion adjuvants induce a strong innate immune response, enhancing antigen presentation both quantitively and qualitatively to generate strong B cell responses and antibody production. They also diversify the reactivity profiles and strengthen the affinities of antibodies against the influenza hemagglutinin, increasing protection across virus clades. In this review, we consider the mechanisms of the enhancement of innate and adaptive immune responses by squalene-based emulsionSE adjuvants and the resulting increase in magnitude and breadth of hemagglutinin-specific B cell responses. We relate observed effects of SE adjuvants and current mechanistic understandings to events in responding lymph nodes. These insights will guide the rational design and optimization of influenza vaccines to provide broad and effective protection.

Heterogeneous Responses of Axillary Lymph Node Metastases to Neoadjuvant Chemotherapy are Common and Depend on Breast Cancer Subtype.

The objective of this study was to analyze heterogeneous responses of axillary lymph node metastasis to neoadjuvant chemotherapy and to determine to what extent they differ between tumor subtypes (TN, HER2+, HR+/HER2-). This retrospective, monocenter study included 72 consecutive, histologically node-positive breast cancers (cT1-4 cN1-3 cM0) diagnosed in the period from January 2015 to December 2016, who had received axillary lymph node dissection following neoadjuvant chemotherapy. All individual lymph node specimens were re-evaluated for the presence of tumor cells and chemotherapy effects to assess their response to neoadjuvant chemotherapy on an individual lymph node level according to the Sataloff classification. Heterogeneous axillary responses to neoadjuvant chemotherapy occurred in 47.2% of the included 72 patients. The partial response rate was significantly higher in HR+/HER2- tumors (74.2%) than in TN (28.6%) and HER2+ tumors (25.0%) (p < 0.001). The presence of at least one negative, completely responding lymph node in the axillary lymph node dissection specimen had a false-negative rate of 48.8% in predicting ypN0. It dropped below 10% if at least four completely responding negative lymph nodes were identified. Our study shows that axillary heterogeneous response rates differ significantly between tumor subtypes.

MTE 16.01 Difficulties of Surgery After Induction of Chemoradiotherapy

Difficulties of surgery after induction chemoradiotherapy (CRT) Surgery after induction therapy can be very challenging. From my own observations, this may particularly be an issue when the patient had nodal involvement at presentation and experienced a good response to the induction treatment (induction CRT or even induction C alone) where fibrosis of the responding lymph nodes may make the hilar vascular mobilization more difficult. The following lines will describe some tips on how to minimize the risks of such resections.

Immunologic and Clinical Responses To a CD20-Targeted Immunocytokine, DI-Leu16-IL2, In Relapsed Non-Hodgkin Lymphoma

BackgroundTumor-targeted antibody-cytokine fusion proteins, known as immunocytokines (ICKs) have potent antitumor activity in several preclinical tumor models. We have engineered an anti-CD20-interleukin 2 (IL-2) ICK based on the Leu16 anti-CD20 antibody with de-immunized variable (V) region and junction between the heavy (H) chain constant region and IL-2 (DI-Leu16-IL2). In-vitro studies of DI-Leu16-IL2 have demonstrated CD20 targeting, IL2 bioactivities, and antibody-dependent cell-mediated cytotoxicity (ADCC) functions. In a SCID mouse model, DI-Leu16-IL2 showed superior anti-lymphoma effects over rituximab or rituximab combined with systemic IL-2. MethodsWe have initiated a pilot trial of DI-Leu16-IL20 in CD20+ B-cell non-Hodgkin lymphoma (NHL) under an IRB-approved protocol (clinicaltrials.gov: NCT00720135). A total of 7 patients have been treated with DI-Leu16-IL20 at 0.5mg/m2. The first 2 cases received DI-Leu16-IL2 by intravenous infusion on days 2, 4 and 23, 25 after peripheral B cell depletion with low-dose rituximab (50mg/m2) on days 1 and 22. Five remaining patients received DI-Leu16-IL2 weekly for 4 weeks (days 2, 9, 16, 23) after B cell depletion with low-dose rituximab on days 1, 8, 15, and/or 22 as necessary, based on serum rituximab levels. Clinical ResponsesThe treatment has been generally well tolerated with the majority of adverse events (AE) grade 1 or 2. Non-DLT grade 3 AE included one case of hyperglycemia, infection (herpes zoster), and prolonged QTc. One patient developed grade 3 hyponatremia, which was the only DLT in the study to date. One patient with diffuse large B cell NHL of the lower extremity achieved a CR after 2 cycles (significant PR after cycle 1; Figure 1). Another patient showed a temporal increase in FDG uptake on PET after cycle 1, which decreased after cycle 2 in 2 of the 3 responding lymph nodes, suggesting a local immunologic reaction with corresponding FDG uptake (immune-mediated FDG flare). A lymph node biopsy demonstrated a dense infiltration of T cells. One patient who had her residual tumor biopsied after DI-Leu16-IL2 treatment demonstrated loss of CD20 expression.Pre-ICK B Cell Depletion and Pharmacologic Data: We have successfully maintained minimal or absent peripheral blood B cells, with a level-guided dosing of low-dose rituximab. The Cmax ranged between 75 and 125 ng/ml following each infusion of DI-Leu16-IL2. The half life was 6-8 hours and did not change significantly between doses. We have detected no anti-DI-Leu16-IL2 antibodies in any of the 7 patients treated on this protocol. [Display omitted] Immunologic ResponsesDI-Leu16-IL2 was highly immunologically active. There was a significant increase in local infiltrating T cells (predominantly CD8 T cells) in lymph node samples taken after DI-Leu16-IL2 in 3 of the 4 cases whose samples were available. Soluble IL2 receptor levels spiked with each DI-Leu16-IL2 infusion, up to 3 times above baseline. Among the cytokines and chemokines we evaluated using the Luminex platform, IL-5, IL-7, IL-10, IP10, IL-15, and IFN-gamma demonstrated high spikes (>2-fold increase) within 12 hours from each DI-Leu16-IL2 infusion, while IL-1, IL-12, MIG, TNF-alpha, IFN-alpha, and GCSF levels had a lesser degree of spikes (<2-fold increase). The data on the CR patient seem to show higher peak levels of IL15, IL7, and IP10, while MCP1, IL5, IL10, and MCP appeared lower compared with others. Flow cytometry-based cellular analyses showed no significant increase in regulatory T cells or myeloid-derived suppressor cells. Peripheral blood T cells were analyzed for TCR CDR3 sequence, and demonstrated oligoclonal T cell expansions, suggestive of antigen-driven T cell expansion. ConclusionThis study demonstrated the clinical and immunologic data consistent with a proof-of-principle that DI-Leu16-IL2 is capable of inducing both local and systemic immune responses at a dose 0.5mg/m2, and was associated with a CR in a patient who had highly refractory NHL. A multi-center phase I trial of subcutaneously administered DI-Leu16-IL2 is currently underway. Disclosures:Gillies: Provenance Biopharmaceuticals Corp (employment and equity ownership), Alopexx Oncology (Consultancy) : Consultancy, Employment, Equity Ownership.

SU‐GG‐J‐151: Intra‐Patient Response Heterogeneity Using FLT PET during Chemotherapy in Canine Subjects with Lymphoma

Purpose: PET is routinely used for treatment response assessment in lymphoma after the completion of therapy. However, characterization of lymph node response heterogeneity during therapy could result in treatment modification to improve outcome. We used PET during chemotherapy to assess intra‐patient response heterogeneity in canine subjects with lymphoma. Method and Materials: Nine dogs with lymphoma were treated with GS‐9219, a novel antineoplastic agent preferentially targeting proliferative lymphoid cells. Using the cellular proliferation marker [18F]FLT, whole‐body PET/CT scans were acquired pre‐treatment and after 1, 3, and 5 chemotherapy cycles. Lymph nodes (∼16/dog) were segmented on CT images and these contours were applied to PET images to extract mean and maximum SUV for each node. Lymph node response was defined as relative change in SUV. For each dog, nodal responses and baseline values were normalized to their respective means. Heterogeneity was measured using the coefficient of variation (CV) and range of nodal responses and baseline values. Results: Intra‐patient heterogeneity of lymph node baseline values and responses were substantial. Within each dog, the average variation of mean SUV baseline values (CV: 30%) was slightly less than that of response (CV: 40%). Similar heterogeneities were measured using maximum SUV. There was no significant correlation between heterogeneities of baseline uptake and response (p=0.3). Individual dogs responded differently to treatment as some responses were very heterogeneous (most heterogeneous, CV: 115%) while others were quite uniform (least heterogeneous, CV: 5%). Response heterogeneity increased throughout therapy with CV of 35% after 1 chemotherapy cycle, 55% after 3 cycles, and 60% after 5 cycles. Heterogeneity trends measured using CV and ranges were consistent. Conclusions: During chemotherapy, there was substantial intra‐patient response heterogeneity (∼40%) in dogs with lymphoma. Early PET assessment of nodal response heterogeneity may identify poorly responding lymph nodes, permit early intervention, and improve outcomes in lymphoma patients.

Mutual Helper Effect in Copulsing of Dendritic Cells With 2 Antigens

To develop an efficient dendritic cell (DC)-based immunotherapy protocol, we examined whether simultaneous pulsing of DCs with a given antigen and a third-party antigen could enhance their antigen presentation capacity. Purified splenic DCs of Balb/c mice were pulsed separately with immunoglobulin G, ovalbumin, conalbumin, P15 peptide of Mycobacterium tuberculosis, and prostate-specific antigen or double combinations of the aforementioned antigens. In some settings, DCs pulsed with 1 antigen were mixed equally with those pulsed with another antigen. Antigen-pulsed DCs were injected into the footpad of syngeneic mice and proliferation of whole, CD4 and CD8 depleted lymph node cells was measured after restimulation with cognate antigen. Antigen-specific production of interferon-gamma (IFNgamma) was tested in culture supernatants. Frequency of responding lymph node cells was determined by IFNgamma enzyme-linked immunosorbent spot assay. Our results showed that copulsing of DCs with 2 unrelated antigens increased the capacity of DCs to induce antigen-specific T-cell proliferation against both antigens up to 16-fold. Injection of 2 populations of DCs each pulsed with a different antigen, increased proliferation of primed T cells significantly as well. Both CD4 and CD8 depleted populations showed vigorous proliferative response in copulsing system. In addition, copulsing of DCs with 2 antigens resulted in higher frequency of antigen-specific responding cells and significantly more IFNgamma production. Our results clearly showed that unrelated peptides and proteins could be used to enhance efficacy of DC-based vaccines and in this system, each antigen served to help the other one, a condition that we termed as "mutual helper effect."

Do the lungs deliver naïve CD4 T cells to draining nodes? (95.13)

Abstract ~80% of lung CD4 T cells express low levels of CD44, high levels of CD62L, CD45RB, CCR7 and CD127 consistent with their being naïve. When Rag2-/- TCR transgenic cells are transferred to syngeneic mice, naïve phenotype transgenic cells are found in the lung. These cells disappear rapidly upon treatment with FTY-720 but are still found in mice treated with anti-CD62L, implying they are part of the recirculating pool but do not require L-selectin to enter the lung. Upon intranasal immunization with low (1 μg) antigen dose, the response is limited to the nodes draining the airways. When these low antigen doses are used, responses are not efficient as shown by the persistence of naïve antigen-specific CD4 T cells in the non-draining nodes. Nonetheless, "naïve" antigen-specific cells are lost from the lung; they reappear as activated cells at about 2.5 days after immunization, a response that is inhibited by FTY720. These results indicate that primary responses occur in the organized draining lymphoid tissue, as would be expected, but imply that the afferent lymphatics may be an important source for the delivery of naive CD4 T cells from tissues to the responding lymph nodes.

Infection of cattle with Theileria parva induces an early CD8 T cell response lacking appropriate effector function

Theileria parva causes an acute lympho-proliferative disease in cattle, which can result in death of susceptible animals within 2–3 weeks of infection. Analyses of the cellular response in the lymph node draining the site of infection demonstrated an early T cell response, with the appearance of large numbers of uninfected lymphoblasts between 6 and 9 days p.i., coinciding with initial detection of parasitised cells. There was a marked increase in the representation of CD8+ T cells and the emergence of a sizable sub-population of CD2− CD8+ α/β T cells during this period. Analysis of T cell receptor β chain variable (TCR BV) gene expression did not reveal any evidence for the involvement of a superantigen in stimulating the response. Responding lymph node cells were found to produce increased quantities of IFNγ and IL-10, and both the CD2+ CD8+ and CD2− CD8+ populations expressed IFNγ transcripts. Purified CD2+ CD8+ cells proliferated when stimulated in vitro with autologous parasitised cells or non-specific mitogens, whereas CD2− CD8+ cells were refractory to these stimuli. In contrast to the parasite-specific cytotoxic activity associated with T cell responses in immune cattle, the responses to primary infection exhibited variable levels of non-specific cytotoxic activity. Stimulation of purified CD2+ CD8+ T cells in vitro with autologous parasitised cells also failed to reveal evidence of specific cytotoxic activity. These findings indicate that primary infection with T. parva induces an aberrant T cell response that lacks appropriate effector activity.

Age‐related changes in the occurrence and characteristics of thymic CD4+ CD25+ T cells in mice

Natural regulatory CD4(+) CD25(+) T cells play an important role in preventing autoimmunity by maintaining self-tolerance. They express CD25 constitutively and are produced in the thymus as a functionally mature T-cell population. Changes in the potential of these cells to regulate the activity of conventional effector lymphocytes may contribute to an increased susceptibility to infection, cancer and age-associated autoimmune diseases. In this study we demonstrated that the thymi of aged mice are populated by a higher percentage of CD4(+) CD25(+) thymocytes than in young animals. The expression of several surface markers (CD69, CD5, CD28, CTLA-4, CD122, FOXP3), usually used to characterize the phenotype of CD4(+) CD25(+) T regulatory cells, was compared between young and aged mice. We also examined the ability of sorted thymus-deriving regulatory T cells of young and aged BALB/c mice to inhibit the proliferation of lymph node lymphocytes activated in vitro. Natural regulatory T cells isolated from the thymi of young mice suppress the proliferation of responder lymph node cells. We demonstrated that thymus-deriving CD4(+) CD25(+) T cells of old mice maintain their potential to suppress the proliferation of activated responder lymphocytes of young mice. However, their potential to inhibit the proliferation of old responder T cells is abrogated. Differences in the occurrence and activity of CD4(+) CD25(+) thymocytes between young and old animals are discussed in relation to the expression of these surface markers.

Recirculating CD4 memory T cells mount rapid secondary responses without major contributions from follicular CD4 effectors and B cells

For weeks after primary immunization with thymus-dependent antigens the responding lymph nodes contain effector CD4 T cells in T zones and germinal centers as well as recirculating memory T cells. Conversely, remote nodes, not exposed to antigen, only receive recirculating memory cells. We assessed whether lymph nodes with follicular effector CD4 T cells in addition to recirculating memory CD4 T cells mount a more rapid secondary response than nodes that only contain recirculating memory cells. Also, the extent to which T cell frequency governs accelerated CD4 T cell recall responses was tested. For this, secondary antibody responses to a superantigen, where the frequency of responding T cells is not increased at the time of challenge, were compared with those to conventional protein antigens. With both types of antigens similar accelerated responses were elicited in the node draining the site of primary immunization and in the contralateral node, not previously exposed to antigen. Thus recirculating memory cells are fully capable of mounting accelerated secondary responses, without the assistance of CD4 effector T cells, and accelerated memory responses are not solely dependent on higher T cell frequencies. Accelerated memory CD4 T cell responses were also seen in B cell-deficient mice.

Intraperitoneal Echinococcus multilocularis infection in C57BL/6 mice affects CD40 and B7 costimulator expression on peritoneal macrophages and impairs peritoneal T cell activation

One of the most important immunopathological consequence of intraperitoneal alveolar echinococcosis (AE) in the mouse is suppression of T cell-mediated immune responses. We investigated whether and how intraperitoneal macrophages (MØs) are, respectively, implicated as antigen-presenting cells (APCs). In a first step we showed that peritoneal MØs from infected mice (AE-MØs) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells. In a subsequent step, AE-MØs as well as naïve MØs (positive control) proved their ability to uptake and process C-Ova fluorescein isthiocyanate (FITC). Furthermore, in comparison with naïve MØs, the surface expression of Ia molecules was up-regulated on AE-MØs at the early stage of infection, suggesting that AE-MØs provide the first signal via the antigen-Ia complex. To study the accessory activity of MØs, AE-MØs obtained at the early and late stages of infection were found to decrease Con A-induced proliferation of peritoneal naïve T cells as well as of AE-sensitized peritoneal T cells, in contrast to stimulation with naïve MØs. The status of accessory molecules was assessed by analysing the expression level of costimulatory molecules on AE-MØs, with naïve MØs as controls. It was found that B7-1 (CD80) and B7-2 (CD86) expression remained unchanged, whereas CD40 was down-regulated and CD54 (= ICAM-1) was slightly up-regulated. In a leucocyte reaction of AE-MØs with naïve or AE-T cells, both types of T cells increased their proliferative response when CD28 - the ligand of B7 receptors - was exposed to anti-CD28 in cultures. Conversely to naïve MØs, pulsing of AE-MØs with agonistic anti-CD40 did not even partially restore their costimulatory activity and failed to increase naïve or AE-T cell proliferation. Neutralizing anti-B7-1, in combination with anti-B7-2, reduced naïve and AE-T cell proliferation, whereas anti-CD40 treatment of naïve MØs increased their proliferative response to Con A. These results point at the key role of B7 receptors as accessory molecules and the necessity of the integrity of CD40-expression by naïve MØs to improve their accessory activity. Taken together, the obstructed presenting-activity of AE-MØs appeared to trigger an unresponsiveness of T cells, contributing to the suppression of their clonal expansion during the chronic phase of AE-infection.

Molecular survival strategies of Echinococcus multilocularis in the murine host

Larval infection with Echinococcus multilocularis starts with the intrahepatic postoncospheral development of a metacestode that—at its mature stage—consists of an inner germinal and an outer laminated layer (GL & LL). In certain cases, an appropriate host immune response may inhibit parasite proliferation. Several lines of evidence obtained in vivo and in vitro indicate the important bio-protective role of the LL. For instance, the LL has been proposed to protect the GL from nitric oxide produced by periparasitic macrophages and dendritic cells, and also to prevent immune recognition by surrounding T cells. On the other hand, the high periparasitic NO production by peritoneal exsudate cells contributes to periparasitic immunosuppression, explaining why iNOS deficienct mice exhibit a significantly lower susceptibility towards experimental infection. The intense periparasitic granulomatous infiltration indicates a strong host–parasite interaction, and the involvement of cellular immunity in control of the metacestode growth kinetics is strongly suggested by experiments carried out in T cell deficient mouse strains. Carbohydrate components of the LL, such as Em2(G11) and Em492, as well as other parasite metabolites yield immunomodulatory effects that allow the parasite to survive in the host. I.e., the IgG response to the Em2(G11)-antigen takes place independently of alpha-beta + CD4 + T cells, and in the absence of interactions between CD40 and CD40 ligand. Such parasite molecules also interfere with antigen presentation and cell activation, leading to a mixed Th1/Th2-type response at the later stage of infection. Furthermore, Em492 and other (not yet published) purified parasite metabolites suppress ConA and antigen-stimulated splenocyte proliferation. Infected mouse macrophages (AE-MØ) as antigen presenting cells (APC) exhibited a reduced ability to present a conventional antigen (chicken ovalbumin, C-Ova) to specific responder lymph node T cells when compared to normal MØ. As AE-MØ fully maintain their capacity to appropriately process antigens, a failure in T cell receptor occupancy by antigen-Ia complex or/and altered co-stimulatory signals can be excluded. Studying the status of accessory molecules implicated in T cell stimulation by MØ, it could be shown that B7-1 (CD80) and B7-2 (CD86) remained unchanged, whereas CD40 was down-regulated and CD54 (=ICAM-1) slightly up-regulated. FACS analysis of peritoneal cells revealed a decrease in the percentage of CD4+ and CD8 + T cells in AE-infected mice. Taken together the obstructed presenting-activity of AE-MØ appeared to trigger an unresponsiveness of T cells leading to the suppression of their clonal expansion during the chronic phase of AE infection. Interesting information on the parasite survival strategy and potential can be obtained upon in vitro and in vivo treatment. Hence, we provided very innovative results by showing that nitazoxanide, and now also, respectively, new modified compounds may represent a useful alternative to albendazole. In the context of chemotherapeutical repression of parasite growth, we searched also for parasite molecules, whose expression levels correlate with the viability and growth activity of E. multilocularis metacestode. Expression levels of 14-3-3 and II/3-10, relatively quantified by realtime reverse transcription-PCR using a housekeeping gene beta-actin, were studied in permissive nu/nu and in low-permissive wild type BALB/c mice. At 2 months p.i., the transcription level of 14-3-3 was significantly higher in parasites actively proliferating in nu/nu mice compared to parasites moderately growing in wild type mice. Immunoblotting experiments confirmed at the protein level that 14-3-3 was over-expressed in parasites derived from nu/nu mice at 2 months p.i. In vitro-treatment of E. multilocularis with an anti-echinococcal drug nitazoxanide for a period of 8 days resulted in a significant decrease of both 14-3-3 and II/3-10 transcription levels, which correlated with the kinetics of a housekeeping gene, beta-actin. This indicates that 14-3-3-exhibits a good potential as a molecular marker to assess viability and growth activity of the parasite.

Immunosuppressive properties of human amniotic membrane for mixed lymphocyte reaction.

The combination of allograft limbal transplantation (ALT) and amniotic membrane transplantation (AMT) has been applied in the treatment of severe ocular surface diseases. The beneficial effect of this combination has been thought to result from possible immunosuppressive ability of amniotic membrane (AM). However, the mechanisms of any such ability remain unknown. In this study, we investigated whether human AM has the ability to suppress allo-reactive T cell responses in vitro. For mixed lymphocyte reaction (MLR), lymphocytes isolated from lymph nodes of C57BL/6 mice (Mls1b, Vbeta6+) were cultured with irradiated splenocytes from DBA/2 mice (Mls1a, Vbeta6-) with or without human AM. For carboxyfluorescein diacetate succinimidyl ester (CFSE) experiments, responder lymph node cells were labelled with a stable intracellular fluorescent dye and cultured with irradiated stimulator cells. The ratio of responder Vbeta6+ T cells was then determined by FACS analysis, and the division profiles of responder Vbeta6+ T cells were analysed by CFSE content. Furthermore, Th1 and Th2 cytokine synthesis by allo-reactive T cells in MLR culture supernatants was determined by enzyme-linked immunosorbent assay (ELISA). Addition of AM to the MLR culture resulted in the significant inhibition of thymidine incorporation compared with control culture lacking AM. The population of responder CD4+Vbeta6+ T cells was significantly reduced in the AM-treated culture in comparison to control. CFSE analysis revealed less division and lower proliferation of responder CD4+Vbeta6+ T cells in cultures with AM than without. In addition, allo-rective T cell synthesis of both Th1 (IL-2 and IFNgamma) and Th2 (IL-6 and IL-10) type cytokine was significantly decreased in the presence of AM. These results indicate that human AM has the ability to suppress allo-reactive T cells in vitro. This inhibitory effect likely contributes to the success of the ALT-AMT combination.

Cellular immunity against Theileria parva and its influence on parasite diversity

Theileria parva, a tick-borne parasite of African cattle, causes a fatal disease known as East Coast fever. Cattle that recover from the disease develop strong parasite-specific MHC -class I-restricted cytotoxic T-lymphocyte responses. Protection can be transferred between immune and naı̈ve calves in the CD 8+T cell fraction emanating from a responding lymph node. In vitro studies suggest that this response requires input from activatedCD 4+T cells. The T parva life cycle involves developmental stages in mammalian and tick hosts and can lead to a number of different endemic scenarios for the disease. These range from a stable situation with high prevalence of herd infection, but low fatality rates, to a low prevalence/high fatality scenario. The impact on endemic stability is an important consideration for the design of vaccine implementation strategies. For subunit vaccines targeted at T parva schizonts, the principal issue in this regard is whether development of the piroplasm stage is blocked by immunity.

A Comparison of Spontaneous Versus Induced Tolerance in an Experimental Model of Rat Hepatic Allograft Transplantation

In some rodent haplotype combinations, spontaneous tolerance (ST) develops after orthotopic liver transplantation (OLT) without any immunosuppression [e.g., Lewis (Lew, RT1 1) into Wistar Furth (WF, RT1 a)] whereas in other combinations vigorous, progressive rejection rapidly leads to the death of the recipients. We (and others) have induced tolerance (IT) in a rejecting strain combination [Dark Agouti (DA, RT1 a) into Lew] by intrathymic inoculation of donor bone marrow cells and 1 cc of antilymphocyte serum (ALS) 7-14 days prior to OLT. We hypothesized that cellular immunity in the two groups of animals was similar. We first compared survival in each group of animals and found that there was no difference in the number of animals surviving >100 days (8/11 vs 16/17, ST vs IT, respectively, P = 0.11). Liver function studies were similar in these animals at 2 and 4 weeks after OLT and comparable to syngeneic Lew into Lew OLT animals, but significantly lower than in the rejecting DA into Lew combination treated with only ALS. Animals that were unresponsive to their allografts demonstrated donor-specific tolerance by the acceptance of donor strain ( n = 4, ST and IT) and rejection of third party ( n = 1 and n = 2, ST and IT groups, respectively) heterotopic heart allografts. One-way mixed lymphocyte cultures (MLC) of peripheral blood lymphocytes against donor and third party antigen were suppressed to donor and third party stimulators versus the MLC of unmanipulated animals. Naive host strain responder lymph node cells and purified T cells demonstrated strong proliferative responses to donor strain antigen in both the ST and IT animals. Peripheral blood lymphocytes expressing donor MHC class I molecules were not detectable by flow cytometry in the ST animals using the monoclonal antibody (mAb) I-1.69 which is directed against Lew MHC class I molecules. There was variable expression (023%) of donor MHC class I molecules on peripheral lymphocytes in IT animals using the mAb C3, directed against DA MHC class I molecules. In summary, animals unresponsive to their liver allografts demonstrate donor-specific tolerance and functionally intact cellular immunity in vivo by acceptance of donor strain and rejection of third party heart allografts. The MHC class II response is suppressed to donor and third party stimulators in vitro in both groups of animals. In conclusion, cellular immunity in rats after liver allografting in the Lew into WF spontaneously tolerant strain combination is similar to that after tolerance induced by intrathymic manipulation in the DA into Lew combination.

Thy.1lowCD3- cells sorted from nylon wool-passed bone marrow cells can augment the H-2 identical but not non-identical cytotoxic T lymphocyte precursors in mixed lymphocyte cultures.

Thy.1lowCD3- cells obtained from nylon wool-passed murine bone marrow (NW-BM) cells by cell sorting did not express CD4, CD8, or T cell receptor-alpha/beta and -gamma/delta on their cell surfaces. An extremely limited number of B10.BR (H-2k) responder lymph node (LN) cells were stimulated with B10.D2 (H-2d) stimulator spleen cells in cultures containing the minimum required dose of rat T cell growth factor (TCGF). In these cultures, the generation of cytotoxic T lymphocytes (CTL) was very low. B10.BR Thy.1lowCD3- NW-BM cells, added to these cultures, could augment the CTL generation vigorously, but neither B10 (H-2b) nor B10.D2 cells could. When B10 LN cells were used as responder cells in these cultures, B10 Thy.1lowCD3- NW-BM cells could augment the CTL generation, but neither B10.BR nor B10.D2 cells could. Similar findings were obtained when Lyt-2+ cells or Thy.1+L3T4- (CTL precursor) cells sorted from spleen cells were used as responder cells. Both elements, rat-TCGF and Thy.1low CD3- NW-BM cells, were essential for this augmentation of the CTL generation in this culture system because neither one alone could augment generation, and rat-TCGF could be replaced by Thy.1+ Lyt-2- helper T (Th) cells sorted from spleen cells. These findings showed that NW-BM cells could augment CTL precursors in a self-major histocompatibility complex (self-MHC)-antigen restricted manner, and further that both NW-BM cells and Th cells had different and independent functions to induce CTL.

Lymphatic mast cell response and effect of compound 48/80 on popliteal lymph node reaction in rats following intracutaneous injection ofStaphylococcus aureus

To investigate the significance of mast cells in the popliteal lymph node during the development of an inflammatory response, rats were inoculated with 12 x 10(7) colony-forming units of Staphylococcus aureus in the hind foot pad. Numerical changes in mast cells were then measured in the corresponding popliteal lymph node. Six days after inoculation, despite the enlargement of the responding lymph node, a marked decrease in granulated mast cell number, relative to the contralateral node, was observed in the cortical and medullary compartments. Popliteal lymph nodes from rats treated with compound 48/80 and then inoculated with S. aureus showed a higher cortical and medullary hypertrophic response and a significant increase in degranulated/weakly basophilic mast cell number in the lymph node tissue. The findings suggest that (1) Staphylococcus aureus induces a reduction in granulated mast cell number in the cortical and medullary compartments of regional lymph nodes; (2) pretreatment with compound 48/80 appears to contribute to the lymphoid cell proliferation and the hypertrophic response of lymph nodes induced by S. aureus; and (3) granulated mast cells have a regulatory role on lymphoid cell proliferation.

Rat Splenocytes Inhibit Antigen-Specific Lymphocyte Proliferation through a Reactive Nitrogen Intermediate (RNI)-Dependent Mechanism and Exhibit Increased RNI Production in Response to IFN-γ

Rat splenocytes inhibited antigen-specific proliferation of primed lymph node cells in vitro. This inhibition resided in the plastic-adherent splenocyte fraction and was radioresistant, suggesting that the effect was due to macrophages. While this suppression was more evident if spleen cells were derived from immunized rats, spleen cells from normal rats were just as suppressive when added to cocultures at higher numbers. Proliferative responses were greatly enhanced in the presence of NG -monomethyl-L-arginine, a specific inhibitor of the nitric oxide synthetic pathway, and significant levels of nitrite (NO-2), a product of this pathway, were detected in culture supernatants in association with suppressed responses, supporting the notion that suppression was mediated by the L-arginine-dependent production of reactive nitrogen intermediates (RNI). When the splenocytes were physically separated from the responding lymph node cell population, high levels of NO-2 were still detected but proliferative responses were no longer inhibited, suggesting that cell proximity or contact is necessary for delivery of the suppresslye signal. Adherent splenocytes cultured alone produced low levels of NO-2. Addition of 1 to 50 U/ml IFN-γ induced a dose-dependent increase in NO-2 production, with the maximal level approximating that found in suppressed cocultures; TNF-α, IL-2, or LPS did not synergize with IFN-γ to enhance NO-2 production. These findings suggest that by activating macrophages to upregulate RNI synthesis, IFN-γ-producing T cells may exert a negative influence over their own proliferation.

Absence of T cell tolerance to pancreatic islet cells.

To examine whether the lack of self-tolerance to beta cells is responsible for the development of type I diabetes in nonobese diabetic (NOD) mice, we attempted to induce T cell responses to cells from the islets of Langerhans. The data show that all NOD mice, irrespective of age, sex, and disease progression, possess islet cell-specific CD4+, MHC class II-restricted T cells. Both primary and secondary proliferative responses to islet cells were readily induced. The activation of T cells required presentation of islet cell Ag by APC in the responding lymph node cell population. Cells from other tissues, e.g., salivary gland, adrenal gland, and spleen, failed to activate autologous T lymphocytes. T cells specific for other Ag did not respond to islet cells, indicating that the proliferation is not the result of nonspecific stimulation by islet cell products. The presence of islet cell-reactive T cells is, however, not unique to NOD mice, because similar T cell reactivity was also demonstrated in non-diabetes-prone mouse strains. Hence, self-tolerance to islet cells appears to be absent. The results indicate a normal occurrence of islet cell-reactive T cells in both diabetes-prone as well as non-diabetes-prone mice. Thus, the lack of tolerance cannot be the initial cause of diabetes, but the activation of such autoreactive T cells may be important for the development of the disease.

Cellular mechanisms of suppression of T lymphocyte proliferation by lung cells in experimental tuberculosis

The ability of interstitial lung cells from mice, infected with Mycobacterium tuberculosis H37Rv, to suppress proliferative responses of immune lymphocytes to mycobacterial (PPD) and unrelated (Staphylococcus aureus cytoplasm) antigens was studied. Two types of suppression were observed: the specific one, which was characteristic of the PPD-response only; and non-specific. The latter was mediated mainly by prostaglandins, since it could be abolished by indomethacin. Both types of suppression depended on the presence of plastic and nylon wool adherent phagocytes from infected lung. Though the depletion of T or B lymphocytes from the lung cell population have not abrogated the suppressive effect, some intercellular interactions were required for antigen-specific suppression, since the presence of nylon wool adherent cells in the population of responder lymph node cells was necessary for its development.