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Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple autoantibody types, some of which are produced by long-lived plasma cells (LLPC). Active SLE generates increased circulating antibody-secreting cells (ASC). Here, we examine the phenotypic, molecular, structural, and functional features of ASC in SLE. Relative to post-vaccination ASC in healthy controls, circulating blood ASC from patients with active SLE are enriched with newly generated mature CD19 - CD138 + ASC, similar to bone marrow LLPC. ASC from patients with SLE displayed morphological features of premature maturation and a transcriptome epigenetically initiated in SLE B cells. ASC from patients with SLE exhibited elevated protein levels of CXCR4, CXCR3 and CD138, along with molecular programs that promote survival. Furthermore, they demonstrate autocrine production of APRIL and IL-10, which contributed to their prolonged in vitro survival. Our work provides insight into the mechanisms of generation, expansion, maturation and survival of SLE ASC., (© 2024. The Author(s).)

Background: SARS-CoV-2 continues to impact human health globally, with airborne transmission being a significant mode of transmission. In addition to tools like vaccination and testing, countermeasures that reduce viral spread in indoor settings are critical. This study aims to assess the efficacy of UV-C light, utilizing the Violett sterilization device, as a countermeasure against airborne transmission of SARS-CoV-2 in the highly susceptible Golden Syrian hamster model., Methods: Two cohorts of naïve hamsters were subjected to airborne transmission from experimentally infected hamsters; one cohort was exposed to air treated with UV-C sterilization, while the other cohort was exposed to untreated air., Results: Treatment of air with UV-C light prevented the airborne transmission of SARS-CoV-2 from the experimentally exposed hamster to naïve hamsters. Notably, this protection was sustained over a multi-day exposure period during peak viral shedding by hamsters., Conclusions: These findings demonstrate the efficacy of the UV-C light to mitigate against airborne SARS-CoV-2 transmission. As variants continue to emerge, UV-C light holds promise as a tool for reducing infections in diverse indoor settings, ranging from healthcare facilities to households. This study reinforces the urgency of implementing innovative methods to reduce airborne disease transmission and safeguard public health against emerging biological threats.

Emerging infectious disease threats are becoming more frequent due to various social, political, and geographical pressures, including increased human-animal contact, global trade, transportation, and changing climate conditions. Since blood products for transfusion are derived from donated blood from the general population, emerging agents spread by blood contact or the transfusion of blood products are also a potential risk. Blood transfusions are essential in treating patients with anemia, blood loss, and other medical conditions. However, these lifesaving procedures can contribute to infectious disease transmission, particularly to vulnerable populations. New methods have been implemented on a global basis for the prevention of transfusion transmissions via plasma, platelets, and whole blood products. Implementing proactive pathogen reduction methods may reduce the likelihood of disease transmission via blood transfusions, even for newly emerging agents whose transmissibility and susceptibility are still being evaluated as they emerge. In this review, we consider the Mirasol PRT system for blood safety, which is based on a photochemical method involving riboflavin and UV light. We provide examples of how emerging threats, such as Ebola, SARS-CoV-2, hepatitis E, mpox and other agents, have been evaluated in real time regarding effectiveness of this method in reducing the likelihood of disease transmission via transfusions.

Background and Objectives: Convalescent plasma (CP) has been embraced as a safe therapeutic option for coronavirus disease 2019 (COVID-19), while other treatments are developed. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is not transmissible by transfusion, but bloodborne pathogens remain a risk in regions with high endemic prevalence of disease. Pathogen reduction can mitigate this risk; thus, the objective of this study was to evaluate the effect of riboflavin and ultraviolet light (R + UV) pathogen reduction technology on the functional properties of COVID-19 CP (CCP)., Materials and Methods: COVID-19 convalescent plasma units (n = 6) from recovered COVID-19 research donors were treated with R + UV. Pre- and post-treatment samples were tested for coagulation factor and immunoglobulin retention. Antibody binding to spike protein receptor-binding domain (RBD), S1 and S2 epitopes of SARS-CoV-2 was assessed by ELISA. Neutralizing antibody (nAb) function was assessed by pseudovirus reporter viral particle neutralization (RVPN) assay and plaque reduction neutralization test (PRNT)., Results: Mean retention of coagulation factors was ≥70%, while retention of immunoglobulins was 100%. Starting nAb titres were low, but PRNT 50 titres did not differ between pre- and post-treatment samples. No statistically significant differences were detected in levels of IgG (P ≥ 0·3665) and IgM (P ≥ 0·1208) antibodies to RBD, S1 and S2 proteins before and after treatment., Conclusion: R + UV PRT effects on coagulation factors were similar to previous reports, but no significant effects were observed on immunoglobulin concentration and antibody function. SARS-CoV-2 nAb function in CCP is conserved following R + UV PRT treatment., (© 2021 The Authors. Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.)

Background: There is a global increase in whole blood usage and at the same time, emerging pathogens give cause for pathogen reduction technology (PRT). The Mirasol PRT has shown promising results for plasma and platelet concentrate products. Treatment of whole blood with subsequent platelet survival and recovery analysis would be of global value., Study Design and Methods: A two-arm, open-label laboratory study was performed with 40 whole blood collections in four groups: non-leukoreduced non-PRT-treated, non-leukoreduced PRT-treated, leukoreduced non-PRT-treated, and leukoreduced PRT-treated. Leukoreduction and/or PRT-treatment was performed on the day of collection, then all WB units were stored at room temperature for 24 h. Sampling was performed after hold-time and after 24-h storage in RT. If PRT-treatment or leukoreduction, samples were also taken subsequently after treatment. Thirteen healthy volunteer blood donors completed the in vivo study per protocol. All WB units were non-leukoreduced and PRT-treated. Radioactive labeling of platelets from RT-stored, PRT-treated whole blood, sampling of subjects, recovery, and survival calculations were performed according to the Biomedical Excellence for Safer Transfusion Collaborative protocol., Results: In vitro characteristics show that PRT-treatment leads to increased levels of hemolysis, potassium, and lactate, while there are decreased levels of glucose, FVIII, and fibrinogen after 24 h of storage. All values are within requirements for WB. In vivo recovery and survival of platelets were 85.4% and 81.3% of untreated fresh control, respectively., Conclusions: PRT-treatment moderately reduces whole blood quality but is well within the limits of international guidelines. Recovery and survival of platelets are satisfactory after Mirasol treatment., (© 2021 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Background: We are developing cancer immunotherapy based on the use of autologous tumor tissue that has been rendered replication-incompetent but maintains phenotype and metabolic activity post-preparation., Aim: The aim of this study was to evaluate safety and tolerance to injection of the inactivated tumor cell and adjuvant preparation (Innocell™) within 24 hours of administration in a pilot study in canine patients with solid organ tumors. Methodology . Three canine patients demonstrating accessible solid organ tumors of various types were assessed in this study. The local site injection was monitored post-treatment. Clinical signs of adverse reactions were monitored for 24 hours post-treatment. Blood samples were taken pre-treatment and at 8 and 24 hours post-treatment for all subjects. One subject provided samples at 7 days post-treatment. All blood samples were analyzed for cytokine content for both immune system-associated and tumor-associated cytokines., Results: No signs of adverse reactions at the site of injection or systemically were observed in the study period. A slight fever and lethargy were reported in one subject by the owner post-vaccination. Immune system-associated cytokine levels in two of the three animals were elevated post-treatment. Tumor-associated cytokine levels in all three subjects declined post-treatment from baseline levels with the effect most prominent in the subject with a non-excised tumor., Conclusion: Subcutaneous injection of the inactivated tumor cells and adjuvant was well tolerated in this pilot study. Cytokine responses observed were in line with the intended use of the treatment in stimulating immune response without adverse clinical observations. Additional evaluation is warranted., Competing Interests: AG, RG, and JW own stock in PhotonPharma, Inc. AG and RG are inventors of the Innocell technology utilized in this study and are named as inventors on associated patents., (Copyright © 2020 Raymond P. Goodrich et al.)

Background and Objective: Severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is a member of the coronavirus family. Coronavirus infections in humans are typically associated with respiratory illnesses; however, viral RNA has been isolated in serum from infected patients. Coronaviruses have been identified as a potential low-risk threat to blood safety. The Mirasol Pathogen Reduction Technology (PRT) System utilizes riboflavin and ultraviolet (UV) light to render blood-borne pathogens noninfectious, while maintaining blood product quality. Here, we report on the efficacy of riboflavin and UV light against the pandemic virus SARS-CoV-2 when tested in both plasma and platelets units., Materials and Methods: Stock SARS-CoV-2 was grown in Vero cells and inoculated into either plasma or platelet units. Those units were then treated with riboflavin and UV light. The infectious titres of SARS-CoV-2 were determined by plaque assay using Vero cells. A total of five (n = 5) plasma and three (n = 3) platelet products were evaluated in this study., Results: In both experiments, the measured titre of SARS-CoV-2 was below the limit of detection following treatment with riboflavin and UV light. The mean log reductions in the viral titres were ≥3·40 and ≥4·53 for the plasma units and platelet units, respectively., Conclusion: Riboflavin and UV light effectively reduced the titre of SARS-CoV-2 in both plasma and platelet products to below the limit of detection in tissue culture. The data suggest that the process would be effective in reducing the theoretical risk of transfusion transmitted SARS-CoV-2., (©The Authors. Vox Sanguinis published by John Wiley & Sons Ltd on behalf of International Society of Blood Transfusion.)

Background: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has recently been identified as the causative agent for Coronavirus Disease 2019 (COVID-19). The ability of this agent to be transmitted by blood transfusion has not been documented, although viral RNA has been detected in serum. Exposure to treatment with riboflavin and ultraviolet light (R + UV) reduces blood-borne pathogens while maintaining blood product quality. Here, we report on the efficacy of R + UV in reducing SARS-CoV-2 infectivity when tested in human plasma and whole blood products., Study Design and Methods: SARS-CoV-2 (isolate USA-WA1/2020) was used to inoculate plasma and whole blood units that then underwent treatment with riboflavin and UV light (Mirasol Pathogen Reduction Technology System, Terumo BCT, Lakewood, CO). The infectious titers of SARS-CoV-2 in the samples before and after R + UV treatment were determined by plaque assay on Vero E6 cells. Each plasma pool (n = 9) underwent R + UV treatment performed in triplicate using individual units of plasma and then repeated using individual whole blood donations (n = 3)., Results: Riboflavin and UV light reduced the infectious titer of SARS-CoV-2 below the limit of detection for plasma products at 60-100% of the recommended energy dose. At the UV light dose recommended by the manufacturer, the mean log reductions in the viral titers were ≥ 4.79 ± 0.15 Logs in plasma and 3.30 ± 0.26 in whole blood units., Conclusion: Riboflavin and UV light effectively reduced the titer of SARS-CoV-2 to the limit of detection in human plasma and by 3.30 ± 0.26 on average in whole blood. Two clades of SARS-CoV-2 have been described and questions remain about whether exposure to one strain confers strong immunity to the other. Pathogen-reduced blood products may be a safer option for critically ill patients with COVID-19, particularly those in high-risk categories., Competing Interests: The authors have declared that no competing interests exist.

Objective: To determine the prevalence of anti-apoptotic cell (anti-AC) antibodies with the 9G4 idiotype (9G4+) and the relationship between this and other known 9G4+ specificities and disease activity in patients with systemic lupus erythematosus (SLE)., Methods: Serum samples from 60 SLE patients and 40 healthy donors were incubated with apoptotic Jurkat cells and assayed by flow cytometry for the binding of 9G4+ antibodies. The samples were also tested for 9G4+ reactivity against naive B cells and total IgG and IgM anti-AC antibody reactivity., Results: The 9G4+ antibodies bound late ACs in sera from a majority of the SLE patients (60%) but in sera from only 2 healthy control subjects. Among samples with global IgM or IgG anti-AC antibodies, those with 9G4+ anti-AC antibodies predominated. Patients with high levels of 9G4+ anti-AC antibodies were more likely to have active disease. This was the case even in patients with IgG anti-AC antibodies or anti-double-stranded DNA antibodies. Patients with lupus nephritis were also more likely to have 9G4+ anti-AC antibodies. While 9G4+ reactivity to ACs often coincided with anti-B cell reactivity, some samples had distinct anti-AC or anti-B cell reactivity., Conclusion: The 9G4+ antibody represents a major species of anti-AC antibody in SLE serum, and this autoreactivity is associated with disease activity. The anti-AC reactivity of 9G4+ antibodies can be separated from the germline VH4-34-encoded anti-B cell autoreactivity. Our results indicate that ACs are an important antigenic source in SLE that positively selects B cells with intrinsic autoreactivity against other self antigens. This selection of 9G4+ B cells by ACs may represent an important step in disease progression., (Copyright © 2013 by the American College of Rheumatology.)

Ectopic or tertiary lymphoid tissues, such as inducible bronchus-associated lymphoid tissue (iBALT), form in nonlymphoid organs after local infection or inflammation. However, the initial events that promote this process remain unknown. Here we show that iBALT formed in mouse lungs as a consequence of pulmonary inflammation during the neonatal period. Although we found CD4(+)CD3(-) lymphoid tissue-inducer cells (LTi cells) in neonatal lungs, particularly after inflammation, iBALT was formed in mice that lacked LTi cells. Instead, we found that interleukin 17 (IL-17) produced by CD4(+) T cells was essential for the formation of iBALT. IL-17 acted by promoting lymphotoxin-α-independent expression of the chemokine CXCL13, which was important for follicle formation. Our results suggest that IL-17-producing T cells are critical for the development of ectopic lymphoid tissues.

The omentum is a site of B1 cell lymphopoiesis and immune responsiveness to T cell-independent antigens. However, it is unknown whether it supports immune responses independently of conventional lymphoid organs. We showed that the omentum collected antigens and cells from the peritoneal cavity and supported T cell-dependent B cell responses, including isotype switching, somatic hypermutation, and limited affinity maturation, despite the lack of identifiable follicular dendritic cells. The omentum also supported CD4+ and CD8+ T cell responses to peritoneal antigens and recruited effector T cells primed in other locations. Unlike conventional lymphoid organs, milky spots in the omentum developed in the absence of lymphoid tissue-inducer cells, but required the chemokine CXCL13. Although the lymphoid architecture of milky spots was disrupted in lymphotoxin-deficient mice, normal architecture was restored by reconstitution with lymphotoxin-sufficient hematopoietic cells. These results indicate that the milky spots of the omentum function as unique secondary lymphoid organs that promote immunity to peritoneal antigens.

Current influenza vaccines elicit Abs to the hemagglutinin and neuraminidase envelope proteins. Due to antigenic drift, these vaccines must be reformulated annually to include the envelope proteins predicted to dominate in the following season. By contrast, vaccination with the conserved nucleoprotein (NP) elicits immunity against multiple serotypes (heterosubtypic immunity). NP vaccination is generally thought to convey protection primarily via CD8 effector mechanisms. However, significant titers of anti-NP Abs are also induced, yet the involvement of Abs in protection has largely been disregarded. To investigate how Ab responses might contribute to heterosubtypic immunity, we vaccinated C57BL/6 mice with soluble rNP. This approach induced high titers of NP-specific serum Ab, but only poorly detectable NP-specific T cell responses. Nevertheless, rNP immunization significantly reduced morbidity and viral titers after influenza challenge. Importantly, Ab-deficient mice were not protected by this vaccination strategy. Furthermore, rNP-immune serum could transfer protection to naive hosts in an Ab-dependent manner. Therefore, Ab to conserved, internal viral proteins, such as NP, provides an unexpected, yet important mechanism of protection against influenza. These results suggest that vaccines designed to elicit optimal heterosubtypic immunity to influenza should promote both Ab and T cell responses to conserved internal proteins.

Immunity to heterosubtypic strains of influenza is thought to be mediated primarily by memory T cells, which recognize epitopes in conserved proteins. However, the involvement of B cells in this process is controversial. We show in this study that influenza-specific memory T cells are insufficient to protect mice against a lethal challenge with a virulent strain of influenza in the absence of B cells. B cells contribute to protection in multiple ways. First, although non-neutralizing Abs by themselves do not provide any protection to challenge infection, they do reduce weight loss, lower viral titers, and promote recovery of mice challenged with a virulent heterosubtypic virus in the presence of memory T cells. Non-neutralizing Abs also facilitate the expansion of responding memory CD8 T cells. Furthermore, in cooperation with memory T cells, naive B cells also promote recovery from infection with a virulent heterosubtypic virus by generating new neutralizing Abs. These data demonstrate that B cells use multiple mechanisms to promote resistance to heterosubtypic strains of influenza and suggest that vaccines that elicit both memory T cells and Abs to conserved epitopes of influenza may be an effective defense against a wide range of influenza serotypes.

CXC chemokine ligand 13 (CXCL13), CC chemokine ligand 21 (CCL21), and CCL19 are constitutively expressed in secondary lymphoid organs, where they control the placement of lymphocytes and dendritic cells. However, these chemokines are also inducibly expressed in the lung after influenza infection. Here we show that, in the absence of spleen and lymph nodes, the expression of homeostatic chemokines in the lung is essential for local B and T cell responses to influenza and for the development and organization of inducible bronchus-associated lymphoid tissue (iBALT). Surprisingly, despite the association between local CXCL13 expression and the formation of ectopic lymphoid tissues, the loss of CXCL13 in the lung had minimal impact on either the development or function of iBALT. In contrast, the loss of CCL19 and CCL21 impaired iBALT formation as well as B and T cell responses. These results demonstrate that the local expression of homeostatic chemokines in nonlymphoid organs, such as the lung, plays an important role in protective immune responses.

Bronchus-associated lymphoid tissue (BALT) was originally described as a mucosal lymphoid organ in the lungs of some species. However, while the lungs of naive mice and humans typically lack BALT, pulmonary infection in mice leads to the development of inducible BALT (iBALT), which is located in peribronchial, perivascular, and interstitial areas throughout the lung. Here we investigated whether iBALT forms in patients with a variety of interstitial lung diseases. We show that while iBALT can be found in the lungs of patients suffering from multiple diseases, well-developed iBALT is most prevalent in patients with pulmonary complications of RA and Sjögren syndrome. In these patients, iBALT consisted of numerous B cell follicles containing germinal centers and follicular dendritic cells. A loosely defined T cell area surrounded the B cell follicles while lymphatics and high endothelial venules were found at the B cell/T cell interface. Increased expression of lymphoid-organizing chemokines, such as CXCL13 and CCL21, as well as molecules involved in the immunopathology of RA, such as B cell-activating factor of the TNF family (BAFF), ICOS ligand, and lymphotoxin, correlated with more well-developed iBALT. Finally, the presence of iBALT correlated with tissue damage in the lungs of RA patients, suggesting that iBALT participates in local RA pathogenesis.

Secondary lymphoid organs (SLOs) promote primary immune responses by recruiting naive lymphocytes and activated APCs. However, their role in the persistence or responsiveness of memory lymphocytes is unclear. We tested whether memory cells were maintained and could respond to challenge in the absence of SLOs. We found that influenza-specific CD8 cells in the lung acquired a memory phenotype, underwent homeostatic proliferation, recirculated through nonlymphoid tissues, and responded to and cleared a challenge infection in the complete absence of SLOs. Similarly, influenza-specific virus-neutralizing antibody was generated and maintained in the absence of SLOs. Inducible bronchus-associated lymphoid tissue (iBALT) was also formed in the lungs of previously infected mice and may provide a niche for the maintenance of memory cells at the local level. These data show that SLOs are dispensable for the maintenance of immunologic memory and directly demonstrate the utility of local tissues, such as iBALT, in secondary immune responses.

It is generally believed that the production of influenza-specific IgG in response to viral infection is dependent on CD4 T cells. However, we previously observed that CD40-deficient mice generate influenza-specific IgG during a primary infection, suggesting that influenza infection may elicit IgG responses independently of CD4 T cell help. In the present study, we tested this hypothesis and show that mice lacking CD40 or CD4 T cells produce detectable titers of influenza-specific IgG and recover from influenza infection in a manner similar to that of normal mice. In contrast, mice completely lacking B cells succumb to influenza infection, despite the presence of large numbers of functional influenza-specific CD8 effector cells in the lungs. Consistent with the characteristics of a T-independent Ab response, long-lived influenza-specific plasma cells are not found in the bone marrow of CD40-/- and class II-/- mice, and influenza-specific IgG titers wane within 60 days postinfection. However, despite the short-lived IgG response, CD40-/- and class II-/- mice are completely protected from challenge infection with the same virus administered within 30 days. This protection is mediated primarily by B cells and Ab, as influenza-immune CD40-/- and class II-/- mice were still resistant to challenge infection when T cells were depleted. These data demonstrate that T cell-independent influenza-specific Ab promotes the resolution of primary influenza infection and helps to prevent reinfection.

Nasal-associated lymphoid tissue (NALT) orchestrates immune responses to Ags in the upper respiratory tract. Unlike other lymphoid organs, NALT develops independently of lymphotoxin-alpha (LTalpha). However, the structure and function of NALT are impaired in Ltalpha(-/-) mice, suggesting a link between LTalpha and chemokine expression. In this study we show that the expression of CXCL13, CCL19, CCL21, and CCL20 is impaired in the NALT of Ltalpha(-/-) mice. We also show that the NALT of Cxcl13(-/-) and plt/plt mice exhibits some, but not all, of the structural and functional defects observed in the NALT of Ltalpha(-/-) mice. Like the NALT of Ltalpha(-/-) mice, the NALT in Cxcl13(-/-) mice lacks follicular dendritic cells, BP3(+) stromal cells, and ERTR7(+) lymphoreticular cells. However, unlike the NALT of Ltalpha(-/-) mice, the NALT of Cxcl13(-/-) mice has peripheral node addressin(+) high endothelial venules (HEVs). In contrast, the NALT of plt/plt mice is nearly normal, with follicular dendritic cells, BP3(+) stromal cells, ERTR7(+) lymphoreticular cells, and peripheral node addressin(+) HEVs. Functionally, germinal center formation and switching to IgA are defective in the NALT of Ltalpha(-/-) and Cxcl13(-/-) mice. In contrast, CD8 T cell responses to influenza are impaired in Ltalpha(-/-) mice and plt/plt mice. Finally, the B and T cell defects in the NALT of Ltalpha(-/-) mice lead to delayed clearance of influenza from the nasal mucosa. Thus, the B and T cell defects in the NALT of Ltalpha(-/-) mice can be attributed to the impaired expression of CXCL13 and CCL19/CCL21, respectively, whereas impaired HEV development is directly due to the loss of LTalpha.

Bronchus-associated lymphoid tissue (BALT) is occasionally found in the lungs of mice and humans; however, its role in respiratory immunity is unknown. Here we show that mice lacking spleen, lymph nodes and Peyer's patches generate unexpectedly robust primary B- and T-cell responses to influenza, which seem to be initiated at sites of induced BALT (iBALT). Areas of iBALT have distinct B-cell follicles and T-cell areas, and support T and B-cell proliferation. The homeostatic chemokines CXCL13 and CCL21 are expressed independently of TNFalpha and lymphotoxin at sites of iBALT formation. In addition, mice with iBALT, but lacking peripheral lymphoid organs, clear influenza infection and survive higher doses of virus than do normal mice, indicating that immune responses generated in iBALT are not only protective, but potentially less pathologic, than systemic immune responses. Thus, iBALT functions as an inducible secondary lymphoid tissue for respiratory immune responses.

Two models have been proposed to explain the requirement for CD40 signaling in CD8 T cell responses. The first model suggests that CD4 T cells activate antigen-presenting cells (APCs) through CD40 signaling (APC licensing). In turn, licensed APCs are able to prime naive CD8 T cells. The second model suggests that CD154-expressing CD4 T cells activate CD40-bearing CD8 T cells directly. Although the requirement for CD40 in APC licensing can be bypassed by inflammatory responses to pathogens that activate APCs directly, the second model predicts that CD8 responses to all antigens will be dependent on CD40 signaling. Here we determined which model applies to CD8 responses to influenza. We demonstrate that optimal CD8 T cell responses to influenza are dependent on CD40 signaling, however both primary and secondary responses to influenza require CD40 expression on non-T cells. Furthermore, CD40-/- CD8 T cells proliferate and differentiate to the same extent as CD40+/+ CD8 T cells in response to influenza, as long as they have equal access to CD40+/+ APCs. Thus, CD4 T cells do not activate influenza-specific CD8 cells directly through CD40 signaling. Instead, these data support the classical model, in which CD4 T cells provide help to CD8 T cells indirectly by activating APCs through CD40.

CD40 is an important costimulatory molecule for B cells as well as dendritic cells, monocytes, and other APCs. The ligand for CD40, CD154, is expressed on activated T cells, NK cells, mast cells, basophils, and even activated B cells. Although both CD40(-/-) and CD154(-/-) mice have impaired ability to isotype switch, form germinal centers, make memory B cells, and produce Ab, it is not entirely clear whether these defects are intrinsic to B cells, to other APCs, or to T cells. Using bone marrow chimeric mice, we investigated whether CD40 or CD154 must be expressed on B cells for optimal B cell responses in vivo. We demonstrate that CD40 expression on B cells is required for the generation of germinal centers, isotype switching, and sustained Ab production, even when other APCs express CD40. In contrast, the expression of CD154 on B cells is not required for the generation of germinal centers, isotype switching, or sustained Ab production. In fact, B cell responses are completely normal when CD154 expression is limited exclusively to Ag-specific T cells. These results suggest that the interaction of CD154 expressed by activated CD4 T cells with CD40 expressed by B cells is the primary pathway necessary to achieve B cell activation and differentiation and that CD154 expression on B cells does not noticeably facilitate B cell activation and differentiation.

Lymphotoxin-alpha(-/-) (LTalpha(-/-)) mice are thought to be unable to generate effective T and B cell responses. This is attributed to the lack of lymph nodes and the disrupted splenic architecture of these mice. However, despite these defects we found that LTalpha(-/-) mice could survive infection with a virulent influenza A virus. LTalpha(-/-) mice and normal wild-type mice infected with influenza A generated similar numbers of influenza-specific CD8 T cells that were able to produce IFN-gamma and kill target cells presenting influenza peptides. Furthermore influenza-infected LTalpha(-/-) mice produced high titers of influenza-specific IgM, IgG, and IgA. However, both CD8 and B cell immune responses were delayed in LTalpha(-/-) mice by 2-3 days. The delayed cellular and humoral immune response was sufficient to mediate viral clearance in LTalpha(-/-) mice that were infected with relatively low doses of influenza virus. However, when LTalpha(-/-) mice were infected with larger doses of influenza, they succumbed to infection before the immune response was initiated. These results demonstrate that neither LTalpha nor constitutively organized lymphoid tissues, such as lymph nodes and spleen, are absolutely required for the generation of effective immunity against the respiratory virus influenza A. However, the presence of LTalpha and/or lymph nodes does accelerate the initiation of immune responses, which leads to protection from larger doses of virus.

Peyer's patch and nasal-associated lymphoid tissue (NALT) are mucosal lymphoid tissues that appear similar in structure and function. Surprisingly, we found that NALT, unlike Peyer's patch, was formed independently of lymphotoxin (LT)alpha. Furthermore, using mice deficient in the retinoic acid receptor-related orphan receptor-gamma, we found that NALT was formed in the absence of CD4+CD3- cells, which are thought to be the embryonic source of LTalpha. However, we also found that NALT of LTalpha-/- animals was disorganized and lymphopenic, suggesting that the organization and recruitment of lymphocytes within NALT remained dependent on LTalpha. Finally, we demonstrated that both the structure and function of NALT were restored in LTalpha-/- animals upon reconstitution with normal bone marrow. These results demonstrate that the organogenesis of NALT occurs through unique mechanisms.