Trapping of naive lymphocytes triggers rapid growth and remodeling of the fibroblast network in reactive murine lymph nodes.

Adaptive immunity is initiated in T-cell zones of secondary lymphoid organs. These zones are organized in a rigid 3D network of fibroblastic reticular cells (FRCs) that are a rich cytokine source. In response to lymph-borne antigens, draining lymph nodes (LNs) expand several folds in size, but the fate and role of the FRC network during immune response is not fully understood. Here we show that T-cell responses are accompanied by the rapid activation and growth of FRCs, leading to an expanded but similarly organized network of T-zone FRCs that maintains its vital function for lymphocyte trafficking and survival. In addition, new FRC-rich environments were observed in the expanded medullary cords. FRCs are activated within hours after the onset of inflammation in the periphery. Surprisingly, FRC expansion depends mainly on trapping of naïve lymphocytes that is induced by both migratory and resident dendritic cells. Inflammatory signals are not required as homeostatic T-cell proliferation was sufficient to trigger FRC expansion. Activated lymphocytes are also dispensable for this process, but can enhance the later growth phase. Thus, this study documents the surprising plasticity as well as the complex regulation of FRC networks allowing the rapid LN hyperplasia that is critical for mounting efficient adaptive immunity.

Detection of micrometastases in sentinel lymph nodes from melanoma patients: direct comparison of multimarker molecular and immunopathological methods.

The technique of sentinel lymph node (SLN) dissection is a reliable predictor of metastatic disease in the lymphatic basin draining the primary melanoma. Reverse transcription-polymerase chain reaction (RT-PCR) is emerging as a highly sensitive technique to detect micrometastases in SLNs, but its specificity has been questioned. A prospective SLN study in melanoma patients was undertaken to compare in detail immunopathological versus molecular detection methods. Sentinel lymphadenectomy was performed on 57 patients, with a total of 71 SLNs analysed. SLNs were cut in slices, which were alternatively subjected to parallel multimarker analysis by microscopy (haematoxylin and eosin and immunohistochemistry for HMB-45, S100, tyrosinase and Melan-A/MART-1) and RT-PCR (for tyrosinase and Melan-A/MART-1). Metastases were detected by both methods in 23% of the SLNs (28% of the patients). The combined use of Melan-A/MART-1 and tyrosinase amplification increased the sensitivity of PCR detection of microscopically proven micrometastases. Of the 55 immunopathologically negative SLNs, 25 were found to be positive on RT-PCR. Notably, eight of these SLNs contained naevi, all of which were positive for tyrosinase and/or Melan-A/MART-1, as detected at both mRNA and protein level. The remaining 41% of the SLNs were negative on both immunohistochemistry and RT-PCR. Analysis of a series of adjacent non-SLNs by RT-PCR confirmed the concept of orderly progression of metastasis. Clinical follow-up showed disease recurrence in 12% of the RT-PCR-positive immunopathology-negative SLNs, indicating that even an extensive immunohistochemical analysis may underestimate the presence of micrometastases. However, molecular analyses, albeit more sensitive, need to be further improved in order to attain acceptable specificity before they can be applied diagnostically.

Fibroblastic reticular cells in lymph nodes regulate the homeostasis of naive T cells.

Interleukin 7 is essential for the survival of naive T lymphocytes. Despite its importance, its cellular source in the periphery remains poorly defined. Here we report a critical function for lymph node access in T cell homeostasis and identify T zone fibroblastic reticular cells in these organs as the main source of interleukin 7. In vitro, T zone fibroblastic reticular cells were able to prevent the death of naive T lymphocytes but not of B lymphocytes by secreting interleukin 7 and the CCR7 ligand CCL19. Using gene-targeted mice, we demonstrate a nonredundant function for CCL19 in T cell homeostasis. Our data suggest that lymph nodes and T zone fibroblastic reticular cells have a key function in naive CD4(+) and CD8(+) T cell homeostasis by providing a limited reservoir of survival factors.

Effect of antiretroviral therapy on apoptosis markers and morphology in peripheral lymph nodes of HIV-infected individuals.

BACKGROUND: CD4+ T cell depletion and destruction and the involution of the lymphoid tissue are hallmarks of HIV infection. Although the underlying mechanisms are still unclear, apoptosis appears to play a central role. The objective of this study was to investigate the effect of antiretroviral therapy on the lymph node tissue, particularly with respect to morphology and apoptosis. PATIENTS AND METHODS: Between 1997 and 1999, two inguinal lymph nodes were excised from 31 previously untreated individuals who were in an early stage of HIV infection, the first one prior to treatment and the second after 16 to 20 months of treatment. Paraffin sections were investigated for lymph node architecture, distribution of cellular and viral markers, apoptosis, and expression of apoptotic key molecules which indirectly reflect apoptotic processes. RESULTS: After 16-20 months of antiretroviral therapy, a significant decrease in highly activated HIV-driven immune response was observed in the lymph node tissue as a marked reduction in follicular hyperplasia, a normalization of the follicular dendritic cell network, a significant increase in the number of CD4+ T cells, and a significant decrease in the number of CD8+ T cells. The expression of several proapoptotic (Fas, TRAIL, and active caspase 3) and antiapoptotic (Bcl-2 and IL-7Ralpha) molecules that were reconstituted in the tissues during therapy resembled their expression in lymph nodes of HIV-negative individuals. Limitations of the study are (a) the lack of untreated patients in the late stages, (b) for ethical reasons, the lack of a control group with untreated patients, and (c) for methodological reasons, the restriction of sequential measurements of apotpotic markers to one-third of the patients. CONCLUSION: Antiretroviral therapy initiated in the early stages in HIV infection may halt the irreversible destruction of the lymph node tissue and may partially normalize apoptotic processes.

Drainage of fluorescent liposomes from the vitreous to cervical lymph nodes via conjunctival lymphatics.

The use of liposomes as carriers for the delivery of biologically active molecules into the eye is of major interest. Indeed, encapsulation of biologically active molecules in liposomes may increase their bioavailability and may induce a sustained release, thus avoiding repeated intraocular injections and reducing side effects. We describe here the fate of rhodamine-conjugated liposomes (Rh-Lip) injected into the vitreous of normal Lewis rats. Twenty-four hours after intravitreal injection fluorescent liposomes were detected in the vitreous, the inner layer of the retina and to a lesser extent in the anterior segment of the eye. In addition, numerous Rh-Lip were also observed in the episclera and conjunctival stroma, in conjunctival lymphatic vessels and cervical lymph nodes (LN) draining the conjunctiva and the eye. In the LN, Rh-Lip were taken up by resident macrophages adjacent to CD4+ and CD8+ T cells. Thus, intravitreal injection of anti-inflammatory drugs loaded in liposomes could modulate the ocular immune microenvironment. In addition the passage of drugs into the cervical LN could alter the immune status of these LN and contribute to the regulation of intraocular inflammation. Our results suggest that this phenomenon should be taken into account to design new therapies based on intraocular drug administration.

Reduced L-selectin (CD62LLow) expression identifies tumor-specific type 1 T cells from lymph nodes draining an autologous tumor cell vaccine.

Reduced expression of CD62L can identify tumor-specific T cells in lymph nodes draining murine tumors. Here, we examined whether this strategy could isolate tumor-specific T cells from vaccinated patients. Tumor vaccine-draining lymph node (TVDLN) T cells of seven patients were separated into populations with reduced (CD62LLow) or high levels of CD62L (CD62LHigh). Effector T cells generated from CD62LLow cells maintained or enriched the autologous tumor-specific type 1 cytokine response compared to unseparated TVDLN T cells in four of four patients showing tumor-specific cytokine secretion. Interestingly, effector T cells generated from CD62LLow or CD62LHigh TVDLN were polarized towards a dominant type 1 or type 2 cytokine profile, respectively. For CD62LLow T cells the type 1 cytokine profile appeared determined prior to culture. Since a tumor-specific type 1 cytokine profile appears critical for mediating anti-tumor activity in vivo, this approach might be used to isolate T cells for adoptive immunotherapy.

Trachea, lung, and tracheobronchial lymph nodes are the major sites where antigen-presenting cells are detected after nasal vaccination of mice with human papillomavirus type 16 virus-like particles.

Vaccination by the nasal route has been successfully used for the induction of immune responses. Either the nasal-associated lymphoid tissue (NALT), the bronchus-associated lymphoid tissue, or lung dendritic cells have been mainly involved. Following nasal vaccination of mice with human papillomavirus type 16 (HPV16) virus-like-particles (VLPs), we have previously shown that interaction of the antigen with the lower respiratory tract was necessary to induce high titers of neutralizing antibodies in genital secretions. However, following a parenteral priming, nasal vaccination with HPV16 VLPs did not require interaction with the lung to induce a mucosal immune response. To evaluate the contribution of the upper and lower respiratory tissues and associated lymph nodes (LN) in the induction of humoral responses against HPV16 VLPs after nasal vaccination, we localized the immune inductive sites and identified the antigen-presenting cells involved using a specific CD4(+) T-cell hybridoma. Our results show that the trachea, the lung, and the tracheobronchial LN were the major sites responsible for the induction of the immune response against HPV16 VLP, while the NALT only played a minor role. Altogether, our data suggest that vaccination strategies aiming to induce efficient immune responses against HPV16 VLP in the female genital tract should target the lower respiratory tract.

Direct presentation of a melanocyte-associated antigen in peripheral lymph nodes induces cytotoxic CD8+ T cells.

Encounter of self-antigens in the periphery by mature T cells induces tolerance in the steady-state. Hence, it is not understood why the same peripheral antigens are also promiscuously expressed in the thymus to mediate central tolerance. Here, we analyzed CD8(+) T-cell tolerance to such an antigen constituted by ovalbumin under the control of the tyrosinase promoter. As expected, endogenous CD8(+) T-cell responses were altered in the periphery of transgenic mice, resulting from promiscuous expression of the self-antigen in mature medullary epithelial cells and deletion of high-affinity T cells in the thymus. In adoptive T-cell transfer experiments, we observed constitutive presentation of the self-antigen in peripheral lymph nodes. Notably, this self-antigen presentation induced persisting cytotoxic cells from high-affinity CD8(+) T-cell precursors. Lymph node resident melanoblasts expressing tyrosinase directly presented the self-antigen to CD8(+) T cells, independently of bone marrow-derived antigen-presenting cells. This peripheral priming was independent of the subcellular localization of the self-antigen, indicating that this mechanism may apply to other melanocyte-associated antigens. Hence, central tolerance by promiscuous expression of peripheral antigens is a mandatory, rather than a superfluous, mechanism to counteract the peripheral priming, at least for self-antigens that can be directly presented in lymph nodes. The peripheral priming by lymph node melanoblasts identified here may constitute an advantage for immunotherapies based on adoptive T-cell transfer.

Enhancing efficacy of anticancer vaccines by targeted delivery to tumor-draining lymph nodes.

The sentinel or tumor-draining lymph node (tdLN) serves as a metastatic niche for many solid tumors and is altered via tumor-derived factors that support tumor progression and metastasis. tdLNs are often removed surgically, and therapeutic vaccines against tumor antigens are typically administered systemically or in non-tumor-associated sites. Although the tdLN is immune-suppressed, it is also antigen experienced through drainage of tumor-associated antigens (TAA), so we asked whether therapeutic vaccines targeting the tdLN would be more or less effective than those targeting the non-tdLN. Using LN-targeting nanoparticle (NP)-conjugate vaccines consisting of TAA-NP and CpG-NP, we compared delivery to the tdLN versus non-tdLN in two different cancer models, E.G7-OVA lymphoma (expressing the nonendogenous TAA ovalbumin) and B16-F10 melanoma. Surprisingly, despite the immune-suppressed state of the tdLN, tdLN-targeting vaccination induced substantially stronger cytotoxic CD8+ T-cell responses, both locally and systemically, than non-tdLN-targeting vaccination, leading to enhanced tumor regression and host survival. This improved tumor regression correlated with a shift in the tumor-infiltrating leukocyte repertoire toward a less suppressive and more immunogenic balance. Nanoparticle coupling of adjuvant and antigen was required for effective tdLN targeting, as nanoparticle coupling dramatically increased the delivery of antigen and adjuvant to LN-resident antigen-presenting cells, thereby increasing therapeutic efficacy. This work highlights the tdLN as a target for cancer immunotherapy and shows how its antigen-experienced but immune-suppressed state can be reprogrammed with a targeted vaccine yielding antitumor immunity.

Absence of ectopic epithelial inclusions in 3,904 axillary lymph nodes examined in sentinel technique.

Intraoperative examination of sentinel axillary lymph nodes can be done by imprint cytology, frozen section, or, most recently, by PCR-based amplification of a cytokeratin signal. Using this technique, benign epithelial inclusions, representing mammary tissue displaced along the milk line, will likely generate a positive PCR signal and lead to a false-positive diagnosis of metastatic disease. To better appreciate the incidence of ectopic epithelial inclusions in axillary lymph nodes, we have performed an autopsy study, examining on 100 μm step sections 3,904 lymph nodes obtained from 160 axillary dissections in 80 patients. The median number of lymph nodes per axilla was 23 (15, 6, and 1 in levels 1, 2, and 3, respectively). A total of 30,450 hematoxylin-eosin stained slides were examined, as well as 8,825 slides immunostained with pan-cytokeratin antibodies. Despite this meticulous work-up, not a single epithelial inclusion was found in this study, suggesting that the incidence of such inclusions is much lower than the assumed 5% reported in the literature.

Changing responsiveness to chemokines allows medullary plasmablasts to leave lymph nodes.

During T cell-dependent antibody responses lymph node B cells differentiate either to plasmablasts that grow in the medullary cords, or to blasts that proliferate in follicles forming germinal centers. Many plasmablasts differentiate to plasma cells locally, but some leave the medullary cords and migrate to downstream lymph nodes. To assess the basis for this migration, changes in the responsiveness of B cells to a range of chemokines have been studied as they differentiate. Naive B cells express high levels of CCR6, CCR7, CXCR4 and CXCR5. When activated B cells grow in follicles the expression of these chemokine receptors and the responsiveness to the respective chemokines is retained. During the extrafollicular response, plasmablast expression of CXCR5 and responsiveness to B-lymphocyte chemoattractant (CXCR5) as well as to secondary lymphoid tissue chemokine (CCR7) and stromal cell-derived factor (SDF)-1 (CXCR4) are lost while a weak response towards the CCR6 chemokine LARC is maintained. Despite losing responsiveness to SDF-1, extrafollicular plasmablasts still express high levels of CXCR4 on the cell surface. These results suggest that the combined loss of chemokine receptor expression and of chemokine responsiveness may be a necessary prerequisite for cells to migrate to the medullary cords and subsequently enter the efferent lymph.

Positive and negative regulation of T cell responses by fibroblastic reticular cells within paracortical regions of lymph nodes.

Fibroblastic reticular cells (FRC) form the structural backbone of the T cell rich zones in secondary lymphoid organs (SLO), but also actively influence the adaptive immune response. They provide a guidance path for immigrating T lymphocytes and dendritic cells (DC) and are the main local source of the cytokines CCL19, CCL21, and IL-7, all of which are thought to positively regulate T cell homeostasis and T cell interactions with DC. Recently, FRC in lymph nodes (LN) were also described to negatively regulate T cell responses in two distinct ways. During homeostasis they express and present a range of peripheral tissue antigens, thereby participating in peripheral tolerance induction of self-reactive CD8(+) T cells. During acute inflammation T cells responding to foreign antigens presented on DC very quickly release pro-inflammatory cytokines such as interferon γ. These cytokines are sensed by FRC which transiently produce nitric oxide (NO) gas dampening the proliferation of neighboring T cells in a non-cognate fashion. In summary, we propose a model in which FRC engage in a bidirectional crosstalk with both DC and T cells to increase the efficiency of the T cell response. However, during an acute response, FRC limit excessive expansion and inflammatory activity of antigen-specific T cells. This negative feedback loop may help to maintain tissue integrity and function during rapid organ growth.