Cell-dependent Antibody Responses Research Articles

Systematic characterization of immunoglobulin loci and deep sequencing of the expressed repertoire in the Atlantic cod (Gadus morhua)

BackgroundThe Atlantic cod is a prolific species in the Atlantic, despite its inconsistent specific antibody response. It presents a peculiar case within vertebrate immunology due to its distinct immune system, characterized by the absence of MHCII antigen presentation pathway, required for T cell-dependent antibody responses. Thorough characterisation of immunoglobulin loci and analysis of the antibody repertoire is necessary to further our understanding of the Atlantic cod’s immune response on a molecular level.ResultsA comprehensive search of the cod genome (gadmor3.0) identified the complete set of IgH genes organized into three sequential translocons on chromosome 2, while IgL genes were located on chromosomes 2 and 5. The Atlantic cod displayed a moderate germline V gene diversity, comprising four V gene families for both IgH and IgL, each with distinct chromosomal locations and organizational structures. 5’RACE sequencing revealed a diverse range of heavy chain CDR3 sequences and relatively limited CDR3 diversity in light chains. The analysis highlighted a differential impact of V-gene germline CDR3 length on receptor CDR3 length between heavy and light chains, underlining different recombination processes.ConclusionsThis study reveals that the Atlantic cod, despite its inconsistent antibody response, maintains a level of immunoglobulin diversity comparable to other fish species. The findings suggest that the extensive recent duplications of kappa light chain genes do not result in increased repertoire diversity. This research provides a comprehensive view of the Atlantic cod's immunoglobulin gene organization and repertoire, necessary for future studies of antibody responses at the molecular level.

Methods for Analysis of Nanoparticle Immunosuppressive Properties.

Adverse drug effects on immune system function represent a significant concern in the pharmaceutical industry, because 10-20% of drug withdrawal from the market is attributed to immunotoxicity. Immunosuppression is one such adverse effect. The traditional immune function test used to estimate materials' immunosuppression is T cell dependent antibody response (TDAR). This method involves a 28-day in vivo study evaluating the animal's antibody titer to a known antigen (Keyhole Limpet Hemocyanin; KLH) with and without challenge. Due to the limited quantities of novel drug candidates, an in vitro method called human lymphocyte activation (HuLA) assay has been developed to substitute the traditional TDAR assay during early preclinical development. In this test, leukocytes isolated from healthy donors vaccinated with the current year's flu vaccine are incubated with Fluzone in the presence or absence of nanoparticles. The antigen-specific lymphocyte proliferation is then measured by ELISA analyzing incorporation of BrdU into DNA of the proliferating cells. Here we describe the experimental procedures for investigating immunosuppressive properties of nanoparticles by both TDAR and HuLA assays, discuss the in vitro-in vivo correlation of these methods, and show a case study using the iron oxide nanoparticle formulation, Feraheme.

The RNA-binding protein hnRNP F is required for the germinal center B cell response

The T cell-dependent (TD) antibody response involves the generation of high affinity, immunoglobulin heavy chain class-switched antibodies that are generated through germinal center (GC) response. This process is controlled by coordinated transcriptional and post-transcriptional gene regulatory mechanisms. RNA-binding proteins (RBPs) have emerged as critical players in post-transcriptional gene regulation. Here we demonstrate that B cell-specific deletion of RBP hnRNP F leads to diminished production of class-switched antibodies with high affinities in response to a TD antigen challenge. B cells deficient in hnRNP F are characterized by defective proliferation and c-Myc upregulation upon antigenic stimulation. Mechanistically, hnRNP F directly binds to the G-tracts of Cd40 pre-mRNA to promote the inclusion of Cd40 exon 6 that encodes its transmembrane domain, thus enabling appropriate CD40 cell surface expression. Furthermore, we find that hnRNP A1 and A2B1 can bind to the same region of Cd40 pre-mRNA but suppress exon 6 inclusion, suggesting that these hnRNPs and hnRNP F might antagonize each-other’s effects on Cd40 splicing. In summary, our study uncovers an important posttranscriptional mechanism regulating the GC response.

Characterizing the cellular and molecular variabilities of peripheral immune cells in healthy recipients of BBIBP-CorV inactivated SARS-CoV-2 vaccine by single-cell RNA sequencing

ABSTRACT Over 3 billion doses of inactivated vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been administered globally. However, our understanding of the immune cell functional transcription and T cell receptor (TCR)/B cell receptor (BCR) repertoire dynamics following inactivated SARS-CoV-2 vaccination remains poorly understood. Here, we performed single-cell RNA and TCR/BCR sequencing on peripheral blood mononuclear cells at four time points after immunization with the inactivated SARS-CoV-2 vaccine BBIBP-CorV. Our analysis revealed an enrichment of monocytes, central memory CD4+ T cells, type 2 helper T cells and memory B cells following vaccination. Single-cell TCR-seq and RNA-seq comminating analysis identified a clonal expansion of CD4+ T cells (but not CD8+ T cells) following a booster vaccination that corresponded to a decrease in the TCR diversity of central memory CD4+ T cells and type 2 helper T cells. Importantly, these TCR repertoire changes and CD4+ T cell differentiation were correlated with the biased VJ gene usage of BCR and the antibody-producing function of B cells post-vaccination. Finally, we compared the functional transcription and repertoire dynamics in immune cells elicited by vaccination and SARS-CoV-2 infection to explore the immune responses under different stimuli. Our data provide novel molecular and cellular evidence for the CD4+ T cell-dependent antibody response induced by inactivated vaccine BBIBP-CorV. This information is urgently needed to develop new prevention and control strategies for SARS-CoV-2 infection. (ClinicalTrials.gov Identifier: NCT04871932).

CD4 T cell responses in persistent Borrelia burgdorferi infection.

Infection of mice with Borrelia burgdorferi (Bb), a tick-transmitted spirochete and the pathogen that causes Lyme disease in humans, triggers CD4 T cell activation in secondary lymphoid tissues, from which they disseminate into various infected tissues. Despite their activation and the appearance of CD4 T cell-dependent antibody responses, Bb establishes persistent infection in natural Bb reservoir hosts in the absence of overt disease, raising the question of the effectiveness of the anti-Bb T cell responses. Reviewing the existing literature, we propose that CD4 T cells might constitute a host cell target of Bb-mediated immune evasion, rendering these cells ineffective in orchestrating effective inflammatory responses and in supporting highly functional Bb-specific antibody induction. Supporting the induction of more effective CD4 T cell responses may help overcome Bb persistence.

Abnormal lower expression of GPR183 in peripheral blood T and B cell subsets of systemic lupus erythematosus patients

G protein-coupled receptor 183 (GPR183) has been indicated to mediate the migration and localisation of immune cells in T cell-dependent antibody responses. Systemic lupus erythematosus (SLE) is a canonical autoimmune disease involving B cell-mediated tolerance destruction and excessive pathogenic autoantibody production, in which multiple GPCRs play a role. To date, there has been no systematic study regarding the expression of GPR183 in lymphocyte subsets of SLE patients. In this research, firstly, we observed the expression trends of GRP183 in various T and B cell subsets in human tonsil tissues. These lymphocyte subsets include CD4+, CD8+, naïve T, effector T, Tfh, activated Tfh, Th1, Th2, Th17, Treg, CD19+CD27-, CD19+CD27+, naïve B, germinal centre B, memory B, and plasma cells. Further, compared with healthy controls (HCs), GPR183 expression levels in above peripheral blood lymphocyte subsets of patients with SLE were reduced overall. The differential expression of GPR183 expression between inactive and active SLE patients indicates that GPR183 expression may be concerned with the disease activity of SLE. This was further confirmed through the strong negative correlation with SLEDAI score and positive correlation with serum complement protein C3, C4 and C1q levels. Further receiver operating characteristic (ROC) curve analysis revealed that GPR183 expression in circulating CD27-IgD+ B cells may be beneficial in distinguishing between inactive and active SLE patients. In addition, type I interferon stimulation could down-regulate the expression of GPR183 in peripheral blood T and B cell subsets. Aberrant expression of GPR183 may provide some novel insights into disease activity prediction and underlying pathogenesis of SLE.

B cells expressing IgM B cell receptors of HIV-1 neutralizing antibodies discriminate antigen affinities by sensing binding association rates.

HIV-1 envelope (Env) proteins designed to induce neutralizing antibody responses allow study of the role of affinities (equilibrium dissociation constant [KD]) and kinetic rates (association/dissociation rates) on B cell antigen recognition. It is unclear whether affinity discrimination during B cell activation is based solely on Env protein binding KD and whether B cells discriminate among proteins of similar affinities that bind with different kinetic rates. Here, we use a panel of Env proteins and Ramos B cell lines expressing immunoglobulin M (IgM) B cell receptors (BCRs) with specificity for CD4-binding-site broadly neutralizing antibodies to study the role of antigen binding kinetic rates on both early (proximal/distal signaling) and late events (BCR/antigen internalization) in B cell activation. Our results support a kinetic model for B cell activation in which Env protein affinity discrimination is based not on overall KD but on sensing of association rate and a threshold antigen-BCR half-life.

Immunotoxicity of Per- and Polyfluoroalkyl Substances: Insights into Short-Chain PFAS Exposure.

Novel per- and polyfluoroalkyl substances (PFAS) were recently identified in drinking water sources throughout North Carolina. These include the perfluoroether acids (PFEAs) perfluoro-2-methoxyacetic acid (PFMOAA), perfluoro-2-methoxypropanoic acid (PFMOPrA), and perfluoro-4-methoxybutanioc acid (PFMOBA). Little toxicological data exist for these PFEAs. Therefore, the present study described signs of toxicity and immunotoxicity following oral exposure. Adult male and female C57BL/6 mice were exposed once/day for 30 days to PFMOAA (0, 0.00025, 0.025, or 2.5 mg/kg), PFMOPrA, or PFMOBA (0, 0.5, 5, or 50 mg/kg). A dose of 7.5 mg/kg of perfluorooctanoic acid (PFOA) was used as a positive control. Terminal body weights, and absolute liver, spleen, or thymus weights did not differ by dose for any compound; exposure to 50 mg/kg of PFMOBA increased relative liver weights in males. Changes in splenic cellularity were observed in males exposed to PFMOPrA and decreased numbers of B and natural killer (NK) cells were observed in males and females exposed to PFMOBA. Exposure did not alter NK cell cytotoxicity or T cell-dependent antibody responses at doses administered. Our results indicate that these “understudied” PFAS have toxicological potential but require additional investigation across endpoints and species, including humans, to understand health effects via drinking water exposure.

Bim establishes the B-cell repertoire from early to late in the immune response.

In T cell-dependent antibody responses, some of the activated B cells differentiate along extrafollicular pathways into low-affinity memory and plasma cells, whereas others are involved in subsequent germinal center (GC) formation in follicular pathways, in which somatic hypermutation and affinity maturation occur. The present study demonstrated that Bim, a proapoptotic BH3-only member of the Bcl-2 family, contributes to the establishment of the B-cell repertoire from early to late stages of immune responses to T cell-dependent antigens. Extrafollicular plasma cells grew in the spleen during the early immune response, but their numbers rapidly declined with the appearance of GC-derived progeny in wild-type mice. By contrast, conditional Bim deficiency in B cells resulted in expansion of extrafollicular IgG1+ antibody-forming cells (AFCs) and this expansion was sustained during the late response, which hampered the formation of GC-derived high-affinity plasma cells in the spleen. Approximately 10% of AFCs in mutant mice contained mutated VH genes; thus, Bim deficiency appears not to impede the selection of high-affinity AFC precursor cells. These results suggest that Bim contributes to the replacement of low-affinity antibody by high-affinity antibody as the immune response progresses.

Recombinant HA-based vaccine outperforms split and subunit vaccines in elicitation of influenza-specific CD4 T cells and CD4 T cell-dependent antibody responses in humans

Although traditional egg-based inactivated influenza vaccines can protect against infection, there have been significant efforts to develop improved formats to overcome disadvantages of this platform. Here, we have assessed human CD4 T cell responses to a traditional egg-based influenza vaccine with recently available cell-derived vaccines and recombinant baculovirus-derived vaccines. Adults were administered either egg-derived Fluzone®, mammalian cell-derived Flucelvax® or recombinant HA (Flublok®). CD4 T cell responses to each HA protein were assessed by cytokine EliSpot and intracellular staining assays. The specificity and magnitude of antibody responses were quantified by ELISA and HAI assays. By all criteria, Flublok vaccine exhibited superior performance in eliciting both CD4 T cell responses and HA-specific antibody responses, whether measured by mean response magnitude or percent of responders. Although the mechanism(s) underlying this advantage is not yet clear, it is likely that both qualitative and quantitative features of the vaccines impact the response.

B cell-intrinsic MHCII expression restricts bacterial dissemination from the gut and maintains metabolic tone

Abstract The expression of major histocompatibility complex class II (MHCII) molecules on the surface of B cells is essential for cognate interactions with T cells and ultimately the generation of high affinity serum antibody responses. Serum antibody responses play an important role in limiting the ability of gut microbes to colonize the systemic compartment. This is important because bacterial dissemination from the gut can have severe physiological consequences ranging from life-threatening septicemia to chronic low grade systemic inflammation. Here we use a conditional knockout mouse model (B-MHCII−/− mice) to determine the physiological implications of loss of T cell dependent serum antibody responses due to B cell-intrinsic ablation of MHCII expression. Loss of MHCII expression on B cells in B-MHCII−/− mice results in a significant reduction in the generation of long-lived plasma cells in the bone marrow and reduced circulating levels of IgG1. Reduced levels of circulating IgG1 is associated with increased levels of bacterial dissemination from the gut into the systemic compartment, as B-MHCII−/− mice have significantly increased titers of anaerobic bacterial colony-forming units in their livers. Importantly, increased bacterial dissemination is associated with an inability of B-MHCII−/− mice to gain weight, suggesting that bacterial dissemination in B-MHCII−/− mice drives the development of a metabolic disorder. Collectively, our data imply that beyond MHCII’s role in infectious immunity, MHCII-mediated antibody responses also play a fundamental role in establishing benign symbiosis with resident microbiota to maintain proper metabolic physiology.

Nonclinical Immunotoxicity Testing in the Pharmaceutical World: The Past, Present, and Future.

An examination for potential direct or indirect adverse effects on the immune system (immunotoxicity) is an established component of nonclinical testing to support safe use of new drugs. Testing recommendations occur in various regulatory guidance documents, especially ICH S8, and these will be presented. Key evaluation usually occurs in toxicology studies with further investigative work a consideration if a positive signal is seen. Expectations around whether findings may occur are related to the type of compound being developed, including a chemically synthesized small molecule, a small molecule oncology drug, a biopharmaceutical, an oligonucleotide, a gene therapy/stem cell product, a vaccine, or reformulation of drugs in liposomes or depots. Examples of immunotoxicity/immunogenicity findings will be discussed for all of these types of compound. Overall, it can be concluded that our main tool for evaluation of potential immunotoxicity/immunogenicity for a new drug still remains standard toxicology study testing with key assessment for effects on clinical pathology and lymphoid organs/tissues (weights and cellularity). Additional evaluation from studies using a T cell-dependent antibody response (TDAR) and lymphocyte phenotyping is also valuable, if needed. Thus, using the tools from the past, it is the role of toxicologists to work with clinical teams now and in the future, to interpret findings from nonclinical testing to possible adverse findings in humans.

First-in-human clinical trial to assess pharmacokinetics, pharmacodynamics, safety, and tolerability of iscalimab, an anti-CD40 monoclonal antibody.

Iscalimab is a fully human, CD40 pathway blocking, nondepleting monoclonal antibody being developed as an immunosuppressive agent. We describe a first-in-human, randomized, double-blind, placebo-controlled study investigating the safety, tolerability, pharmacokinetics, and pharmacodynamics of iscalimab in healthy subjects and rheumatoid arthritis patients. Healthy subjects (n=56) received single doses of intravenous iscalimab (0.03, 0.1, 0.3, 1, or 3mg/kg), or subcutaneous iscalimab (3mg/kg), or placebo. Rheumatoid arthritis patients (n=20) received single doses of intravenous iscalimab (10 or 30mg/kg) or placebo. Iscalimab exhibited target-mediated drug disposition resulting in dose-dependent and nonlinear pharmacokinetics. Complete (≥90%) CD40 receptor occupancy on whole blood B cells was observed at plasma concentrations >0.3-0.4µg/mL. In subjects receiving 3mg/kg iscalimab, antibody responses to keyhole limpet hemocyanin were transiently suppressed. CD40 occupancy by iscalimab prevented ex vivo human rCD154-induced expression of CD69 on B cells in whole blood. All doses were generally safe and well tolerated, with no clinically relevant changes in any safety parameters, including no evidence of thromboembolic events. Iscalimab appears to be a promising blocker of the CD40-CD154 costimulatory pathway with potential use in transplantation and other autoimmune diseases.

Nonclinical Immunotoxicity Testing in the Pharmaceutical World: The Past, Present, and Future.

An examination for potential direct or indirect adverse effects on the immune system (immunotoxicity) is an established component of nonclinical testing to support safe use of new drugs. Testing recommendations occur in various regulatory guidance documents, especially ICH S8, and these will be presented. Key evaluation usually occurs in toxicology studies with further investigative work a consideration if a positive signal is seen. Expectations around whether findings may occur are related to the type of compound being developed, including a chemically synthesized small molecule, a small molecule oncology drug, a biopharmaceutical, an oligonucleotide, a gene therapy/stem cell product, a vaccine, or reformulation of drugs in liposomes or depots. Examples of immunotoxicity/immunogenicity findings will be discussed for all of these types of compound. Overall, it can be concluded that our main tool for evaluation of potential immunotoxicity/immunogenicity for a new drug still remains standard toxicology study testing with key assessment for effects on clinical pathology and lymphoid organs/tissues (weights and cellularity). Additional evaluation from studies using a T cell-dependent antibody response (TDAR) and lymphocyte phenotyping is also valuable, if needed. Thus, using the tools from the past, it is the role of toxicologists to work with clinical teams now and in the future, to interpret findings from nonclinical testing to possible adverse findings in humans.

Human B Cells Engage the NCK/PI3K/RAC1 Axis to Internalize Large Particles via the IgM-BCR.

Growing evidence indicate that large antigen-containing particles induce potent T cell-dependent high-affinity antibody responses. These responses require large particle internalization after recognition by the B cell receptor (BCR) on B cells. However, the molecular mechanisms governing BCR-mediated internalization remain unclear. Here we use a high-throughput quantitative image analysis approach to discriminate between B cell particle binding and internalization. We systematically show, using small molecule inhibitors, that human B cells require a SYK-dependent IgM-BCR signaling transduction via PI3K to efficiently internalize large anti-IgM-coated particles. IgM-BCR-mediated activation of PI3K involves both the adaptor protein NCK and the co-receptor CD19. Interestingly, we here reveal a strong NCK-dependence without profound requirement of the co-receptor CD19 in B cell responses to large particles. Furthermore, we demonstrate that the IgM-BCR/NCK signaling event facilitates RAC1 activation to promote actin cytoskeleton remodeling necessary for particle engulfment. Thus, we establish NCK/PI3K/RAC1 as an attractive IgM-BCR signaling axis for biological intervention to prevent undesired antibody responses to large particles.

Peripheral follicular helper T cells in acute viral diseases: a perspective on dengue.

Follicular helper T cells (TFH) are a predominant subset of CD4+ T cells specialized in providing help to B cells in germinal centers and necessary to generate T cell-dependent antibody responses. Peripheral TFH (pTFH) are the counterpart of TFH found in the circulation, which resemble TFH in many aspects of their phenotype and function. The CD4+ pTFH subset has received a lot of interest recently because they are easy to access and have the potential to serve as a biomarker for long-lasting humoral immunity. This review will discuss recent findings of pTFH in human acute viral diseases with a focus on dengue infection.

Strain differences in histopathological features of lymphoid tissues of SD and F344 rats in a T cell-dependent antibody response assay of cyclophosphamide.

When conducting histopathological evaluation of lymphoid tissues, it is necessary to know the variability and strain differences in histological features of different sites of lymphoid tissues. To investigate in detail the variability of lymphoid tissues and strain differences of control rats as well as those of immune reactivity and sensitivity to immunosuppression, we performed a histopathological analysis of various lymphoid tissues in conjunction with the evaluation of immune function in a T cell-dependent antibody response (TDAR) assay with cyclophosphamide (CP) in Sprague Dawley (SD) and F344 rats. Six-week-old male SD and F344 rats were orally treated with CP at 0 (control) or 4 mg/kg/day for 28 days; keyhole limpet hemocyanin (KLH) was introduced intravenously on Days 14 and 23, and the serum concentrations of anti-KLH antibodies were measured. HE staining and immunohistochemistry for T-cell (CD3) and B-cell (CD45RA) markers were performed using tissues from the spleen, thymus, and various lymph nodes. In CP-treated rats of both strains, decreased concentrations of anti-KLH antibodies were observed. Histopathological analysis revealed decreased lymphocytes mainly in the B-cell area, and these changes induced by CP treatment were more prominent in the F344 rats than in the SD rats. The present study also demonstrated that some of the lymphoid tissues of the control F344 rats were less developed than those of the control SD rats, suggesting that F344 rats might be easily affected by CP-induced immunosuppression. This information concerning rat strain differences in lymphoid tissues will be useful in histopathological evaluation for drug-induced immunotoxicity.

Complement Regulation and Immune Evasion by Hepatitis C Virus.

A prominent role for complement has been identified in the linkage of innate and adaptive immunity. The liver is the main source of complement and hepatocytes are the primary sites for synthesis of complement components in vivo. We have discovered that hepatitis C virus (HCV) impairs C4 and C3 synthesis. Liver damage may diminish capacity of complement synthesis in patients. However, we observed that the changes in measured complement components in chronically HCV infected patients do not correlate with liver fibrosis or rheumatoid factor present in the blood, serum albumin, or alkaline phosphatase levels. Complement component C3 is of critical importance in B cell activation and T cell-dependent antibody responses. C3 activity is required for optimal expansion of CD8+T cells during a systemic viral infection. Deficiencies in complement may predispose patients to infections via ineffective opsonization, and defects in lytic activity via membrane attack complex. Interestingly, C9 is significantly reduced at the mRNA level in chronically HCV infected liver biopsy specimens, while many hepatocyte derived complement components (C6, C8, Factor B, MASP1, and MBL) and unrelated genes remain mostly unaffected. This implies an HCV specific effect, not a global effect from liver disease.

Role of TRAFs in Signaling Pathways Controlling T Follicular Helper Cell Differentiation and T Cell-Dependent Antibody Responses.

Follicular helper T (TFH) cells represent a highly specialized CD4+ T cell subpopulation that supports the generation of germinal centers (GC) and provides B cells with critical signals promoting antibody class switching, generation of high affinity antibodies, and memory formation. TFH cells are characterized by the expression of the chemokine receptor CXCR5, the transcription factor Bcl-6, costimulatory molecules ICOS, and PD-1, and the production of cytokine IL-21. The acquisition of a TFH phenotype is a complex and multistep process that involves signals received through engagement of the TCR along with a multitude of costimulatory molecules and cytokines receptors. Members of the Tumor necrosis factor Receptor Associated Factors (TRAF) represent one of the major classes of signaling mediators involved in the differentiation and functions of TFH cells. TRAF molecules are the canonical adaptor molecules that physically interact with members of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) and actively modulate their downstream signaling cascades through their adaptor function and/or E3 ubiquitin ligase activity. OX-40, GITR, and 4-1BB are the TRAF-dependent TNFRSF members that have been implicated in the differentiation and functions of TFH cells. On the other hand, emerging data demonstrate that TRAF proteins also participate in signaling from the TCR and CD28, which deliver critical signals leading to the differentiation of TFH cells. More intriguingly, we recently showed that the cytoplasmic tail of ICOS contains a conserved TANK-binding kinase 1 (TBK1)-binding motif that is shared with TBK1-binding TRAF proteins. The presence of this TRAF-mimicking signaling module downstream of ICOS is required to mediate the maturation step during TFH differentiation. In addition, JAK-STAT pathways emanating from IL-2, IL-6, IL-21, and IL-27 cytokine receptors affect TFH development, and crosstalk between TRAF-mediated pathways and the JAK-STAT pathways can contribute to generate integrated signals required to drive and sustain TFH differentiation. In this review, we will introduce the molecular interactions and the major signaling pathways controlling the differentiation of TFH cells. In each case, we will highlight the contributions of TRAF proteins to these signaling pathways. Finally, we will discuss the role of individual TRAF proteins in the regulation of T cell-dependent humoral responses.

Abstract A76: Microenginered human lymphoid tissue on chip

Abstract Human immune responses are markedly different from those of rodents or non-human primates. A poignant example of this unmet need is the immunotherapy TGN1412 which caused an unpredicted cytokine storm in 6 patients. Immunotherapy is designed in animal models and then tested on human immune cells in the blood (the least invasive and most accessible resource). However, blood immune cells are significantly different from tissue resident immune cells i.e. immune cells in the tumor, lymph node and other areas of the body. Blood and tissues have different types and proportions of cells, mechanical forces, extracellular matrix (ECM) and resulting biological interactions. One of the reasons of the TGN1412 cytokine storm was that tissue resident immune cells are hypersensitive to the therapy. Organ-on-chip studies of the immune system have the potential to dramatically streamline clinical trials and enable personalized medicine. To model human tissue resident immunity without relying on animal models, we are using the design principles from the human lymph node and organs-on-chip technology to reverse engineer a prototype of human lymphoid tissue on chip with human blood. We have developed a combination of matrix and cellular components to induce formation of lymphocyte organoids in 3D culture reminiscent of organized lymphoid tissue or follicles usually found in secondary and tertiary lymphoid organs. T and B cell functions are simulated in this model as evidenced by cytokine and immunoglobulin M production in response to heat killed formalin fixed Staph aureus Cowan I (SAC), commonly used to induce a T cell-dependent antibody response. Further organoid formation correlates with increasing levels of CXCL13, a cytokine known to be important in the organization of follicles. Interestingly, the 3D lymphocyte organoids express the coinhibitory receptor CTLA-4, known to be expressed in follicular T cells. As expected of lymphoid follicles, activation by SAC causes these organoids to expand and proliferate. We also present preliminary studies optimizing the perfusion of the lymphocyte parenchyma with microengineered channels lined with lymphatic and vascular endothelial cells to model antigen delivery and immune cell trafficking. These devices will enable near real-time assessment of secreted factors, cell trafficking and high-resolution imaging of dynamic cellular interactions that occur in the human lymph node, and hence, they should prove invaluable in studying human cancer response to anti-cancer immunotherapies in the future. As proof-of-concept, we will compare several clinically approved immunotherapies as well as TGN1412 to test the differences between the response of the engineered lymphoid tissue as compared to standard low density cultures as well as high density cultures. Our preliminary data strongly supports potential applications in understanding basic mechanistic human immunology as well as using the lymphoid tissue on chip for development of immunotherapeutics. Citation Format: Girija Goyal, Jaclyn Long, Donald E. Ingber. Microenginered human lymphoid tissue on chip [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A76.