A major barrier in the use of humanized mice as models of HIV-1 (HIV) infection is the inadequate generation of virus-specific antibody responses. Humanized DRAGA (hDRAGA) mice generate antigen-specific class switched antibodies to several pathogens, but whether they do so in HIV infection and the extent to which their secondary lymphoid tissues (sLT) support germinal center responses is unknown. hDRAGA mice were evaluated for their ability to support HIV replication, generate virus-specific antibody responses, develop splenocyte subsets, and organize sLT architecture. hDRAGA mice supported persistent HIV replication and developed modest levels of gp41-specific human IgM and IgG. Spleens from uninfected and HIV infected hDRAGA mice contained differentiated B and CD4+ T cell subsets including germinal center (GC) B cells and T follicular helper cells (TFH); relative expansions of TFH and CD8+ T cells, but not GC B cells, occurred in HIV-infected hDRAGA mice compared to uninfected animals. Immunofluorescent staining of spleen and mesenteric lymph node sections demonstrated atypical morphology. Most CD4+ and CD8+ T cells resided within CD20hi areas. CD20hi areas lacked canonical germinal centers, as defined by staining for IgD-Ki67+cells. No human follicular dendritic cells (FDC) were detected. Mouse FDC were distributed broadly throughout both CD20hi and CD20lo regions of sLT. HIV RNA particles were detected by in situ hybridization within CD20+ areas and some co-localized with mouse FDC. Viral RNA+ cells were more concentrated within CD20hi compared to CD20lo areas of sLT, but differences were diminished in spleen and eliminated in mesenteric lymph nodes when adjusted for CD4+ cell frequency. Thus, hDRAGA mice recapitulated multiple aspects of HIV pathogenesis including HIV replication, relative expansions in TFH and CD8+ T cells, and modest HIV-specific antibody production. Nevertheless, classical germinal center morphology in sLT was not observed, which may account for the inefficient expansion of GC B cells and generation of low titer human antibody responses to HIV-1 in this model.

We have engineered a Human Immune System (HIS)-reconstituted mouse strain (DRAGA mouse: HLA-A2. HLA-DR4. Rag1 KO. IL-2Rγc KO. NOD) in which the murine immune system has been replaced by a long-term, functional HIS via infusion of CD34+ hematopoietic stem cells (HSC) from cord blood. Herein, we report that the DRAGA mice can sustain inducible and transmissible H1N1 and H3N2 influenza A viral (IAV) infections. DRAGA female mice were significantly more resilient than the males to the H3N2/Aichi infection, but not to H3N2/Hong Kong, H3N2/Victoria, or H1N1/PR8 sub-lethal infections. Consistently associated with large pulmonary hemorrhagic areas, both human and murine Factor 8 mRNA transcripts were undetectable in the damaged lung tissues but not in livers of DRAGA mice advancing to severe H1N1/PR8 infection. Infected DRAGA mice mounted a neutralizing anti-viral antibody response and developed lung-resident CD103 T cells. These results indicate that the DRAGA mouse model for IAV infections can more closely approximate the human lung pathology and anti-viral immune responses compared to non-HIS mice. This mouse model may also allow further investigations into gender-based resilience to IAV infections, and may potentially be used to evaluate the efficacy of IAV vaccine regimens for humans.

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Evaluation of sepsis-induced immunoparalysis has highlighted how decreased lymphocyte number/function contribute to worsened infection/cancer. Yet, an interesting contrast exists with autoimmune disease development, wherein diminishing pathogenic effectors may benefit the post-septic host. Within this framework, the impact of cecal ligation and puncture (CLP)-induced sepsis on the development of experimental autoimmune encephalomyelitis (EAE) was explored. Notably, CLP mice have delayed onset and reduced disease severity, relative to sham mice. Reduction in disease severity was associated with reduced number, but not function, of autoantigen (MOG)-specific pathogenic CD4 T cells in the CNS during disease and draining lymph node during priming. Numerical deficits of CD4 T cell effectors are associated with the loss of MOG-specific naive precursors. Critically, transfer of MOG-TCR transgenic (2D2) CD4 T cells after, but not before, CLP led to EAE disease equivalent to sham mice. Thus, broad impairment of antigenic responses, including autoantigens, is a hallmark of sepsis-induced immunoparalysis. eLife digest Sepsis is a life-threatening condition that can happen when the immune system overreacts to an infection and begins to damage tissues and organs in the body. It causes an extreme immune reaction called a cytokine storm, where the body releases uncontrolled levels of cytokines, proteins that are involved in coordinating the body's response to infections. This in turn activates more immune cells, resulting in hyperinflammation. People who survive sepsis may have long-lasing impairments in their immune system that may leave them more vulnerable to infections or cancer. But scientists do not know exactly what causes these lasting immune problems or how to treat them. The fact that people are susceptible to cancer and infection after sepsis may offer a clue. It may suggest that the immune system is not able to attack bacteria or cancer cells. One way to explore this clue would be to test the effects of sepsis on autoimmune diseases, which cause the immune system to attack the body's own cells. For example, in the autoimmune disease multiple sclerosis, the immune system attacks and destroys cells in the nervous system. If autoimmune disease is reduced after sepsis, it would suggest the cell-destroying abilities of the immune system are lessened. Using this approach, Jensen, Jensen et al. show that sepsis reduces the number of certain immune cells, called CD4 T cells, which are are responsible for an autoimmune attack of the central nervous system. In the experiments, mice that survived sepsis were evaluated for their ability to develop a multiple sclerosis-like disease. Mice that survived sepsis developed less severe or no autoimmune disease. After sepsis, these animals also had fewer CD4 T cells. However, when these immune cells were reinstated, the autoimmune disease emerged. The experiments help explain some of the immune system changes that occur after sepsis. Jensen, Jensen et al. suggest that rather than being completely detrimental, these changes may help to block harmful autoimmune responses. The experiments may also hint at new ways to combat autoimmune diseases by trying to replicate some of the immune-suppressing effects of sepsis. Studying the effect of sepsis on other autoimmune diseases in mice might provide more clues. Introduction Sepsis poses both a significant health concern, affecting 1.7 million and killing 270,000 Americans yearly, and economic burden (> $20 billion annually) (CDC, 2020). Sepsis is characterized by cytokine storm, a maladaptive response to a mismanaged infection, that is comprised of both pro- and anti-inflammatory cytokines. Although the acute cytokine storm is a grave situation, patients who survive a septic event are susceptible to further complications with increased susceptibility to unrelated secondary infection, increased viral reactivation, and decreased 5-year survival relative to non-septic patients (Dombrovskiy et al., 2007; Donnelly et al., 2015; Gaieski et al., 2013; Kutza et al., 1998; Walton et al., 2014). These factors have been attributed to the sepsis-induced lymphopenic state and functional deficits of the surviving cells (Hotchkiss et al., 2006; Hotchkiss et al., 2005; Hotchkiss et al., 2001). This state of immunoparalysis is so profound that the majority of sepsis-associated mortality has shifted to be late deaths following secondary infections or other morbid conditions as a result of immunologic impairment (Delano and Ward, 2016). Therefore, emphasis has been strongly shifted toward determining the mechanisms by which sepsis impairs the immune response to subsequent infection or cancer (Chen et al., 2019; Condotta et al., 2015; Condotta et al., 2013; Danahy et al., 2019a; Jensen et al., 2018b). Yet, this focus on the sepsis-induced loss of beneficial host responses has come at the expense of understanding how sepsis may influence other subsequent maladaptive immune responses. As opposed to infection/cancer, wherein effectors promote disease control, autoantigen-specific effectors promote disease and are detrimental to the host. Multiple sclerosis (MS) is a chronic neuroinflammatory disease of the CNS and is potentially the most common non-traumatic cause of CNS disability in young adults (Compston and Coles, 2002). MS poses a major personal and economic burden; it effects mostly women and on average presents at age 30, an age crucial for family planning (Fox, 2004). The etiology of MS involves a complex interaction between genetic and environmental components, resulting in a clinical presentation including, but not limited to, sensory, motor, and/or cognitive deficits (Dendrou et al., 2015; Freedman et al., 2018). MS is thought to result from autoreactive CD4 T cell responses to the myelin antigens in the CNS followed by inflammatory cellular infiltration, demyelination, and neurodegeneration. In experimental autoimmune encephalomyelitis (EAE), a murine model of MS, this role for CD4 T cells is well-established and induced by immunization against myelin antigens (Stinissen et al., 1997). While there is a dearth of knowledge regarding the impact of sepsis on autoimmunity, descriptions of how sepsis influences specific cell subsets can provide insight into how autoimmune disease may be influenced. With regard to the critical role for CD4 T cells in EAE disease, the sepsis-induced lymphopenic state is known to impact both naive and memory CD4 T cells (Cabrera-Perez et al., 2016; Cabrera-Perez et al., 2015; Chen et al., 2017; Jensen et al., 2018a; Sjaastad et al., 2020b). In particular, sepsis can diminish the number of naive CD4 T cell precursors subsequently limiting the number of cells capable of responding to a given antigen (Cabrera-Perez et al., 2015; Martin et al., 2020). Additionally, sepsis can impair the effector function of surviving T cells further limiting the capacity of cells to mount an antigen-specific response (Mohr et al., 2012; Pötschke et al., 2013; Sjaastad et al., 2018). Sepsis can also impair antigen-specific T cell responses through various cell extrinsic factors, including diminished number/function of dendritic cells (DCs) and diminished capacity of endothelial cells to promote chemotaxis (Danahy et al., 2017; Strother et al., 2016). Cumulatively, these findings suggest that, while detrimental to control of pathogens/cancer, sepsis may alternately diminish a host's capacity to develop autoimmunity. Herein, we demonstrate that following cecal ligation and puncture (CLP), mice have diminished EAE disease severity. Autoantigen-specific effector CD4 T cells were reduced in CLP hosts with EAE due to a loss of their naive precursors, which was the determining factor in the impediment of EAE disease. These data further define how the sepsis-induced immunoparalysis reduces host capacity to generate antigen-specific responses, regardless of whether it is a foreign antigen or an endogenous autoantigen. Results Sepsis reduces EAE disease severity and the number of CNS infiltrating pathogenic effector CD4 T cells The influence of the immunoparalysis phase of sepsis, which follows the resolution of the cytokine storm, on the ability of the host to develop autoimmunity is undefined. To address this relationship, we employed well-established models of polymicrobial sepsis and inducible autoimmunity, CLP and EAE, respectively. CLP effectively mimics the pathophysiology of acute peritonitis and corresponding septic inflammation, with the capacity to modulate disease severity through adjustment of surgical parameters (Dejager et al., 2011; Sjaastad et al., 2020a). Likewise, EAE presents an ideal model system to study the impact of sepsis on the development of autoimmune disease since it is a robust, inducible system allowing for manipulations prior to disease initiation (Stinissen et al., 1997). CLP, sham, and non-surgery mice were immunized with MOG35-55 peptide 5 days post-surgery, a time when the cytokine storm has resolved (Danahy et al., 2019b), to induce EAE and disease progression was monitored (Figure 1a). Of note: unless otherwise stated, experimental timepoints are defined with respect to EAE immunization. Importantly, while sham and no surgery control groups of mice developed robust EAE clinical disease, CLP survivors (day 5 CLP group) had substantially diminished disease severity (Figure 1b,c) and delayed disease onset (Figure 1d). Further, CLP hosts do not achieve similar disease severity even when disease course is prolonged (40 days) (Figure 1—figure supplement 1) indicating that diminished disease is not due to delayed disease onset. Figure 1 with 3 supplements see all Download asset Open asset Sepsis ablates EAE disease and reduces CNS pathology. (A) Experimental design: C57Bl/6 mice were either left naive or underwent sham or CLP surgery. All groups were immunized s.c. with MOG35-55 emulsified in complete Freunds adjuvant (CFA) on both flanks 5 days after surgery and given pertussis toxin (PTX) i.p. on the day of immunization as well as 2 days later to induce EAE. An additional group of mice that underwent CLP 25 days prior were also immunized on the same day. Disease onset and severity were monitored over the subsequent 14 days. (B) Average EAE disease score following EAE induction for mice that had either no surgery (gray), Sham surgery (black), D5 CLP surgery (white), or D25 CLP surgery (red). (C) Area under the curve (AUC) of disease scores in panel B following EAE induction. (D) Time to onset of first EAE symptoms following EAE induction in no surgery (gray), Sham surgery (black), D5 CLP surgery (dashed), and D25 CLP (red) mouse groups. (E) Representative photomicrographs of the lumbar spine from mice with EAE that underwent either sham or D5 CLP surgery. Thick arrows indicate areas of inflammation that was scored on H and E stained slides. Luxol fast blue (LFB) stains were also performed and showed subtle demyelination and axonal sheath swelling in sham mice (red box). Vacuolation is indicated in the inserts by the thin arrows. Bars = 200 µm (inset bars = 50 µm). Mean H and E inflammation score of the (F) spinal cord and (G) meninges of mice 15 days-post EAE induction. Data are representative from three independent experiments with 8–12 mice per group. D25 CLP data are from a single experiment. *p<0.05. Error bars represent the standard error of the mean. Figure 1—source data 1 Source data for Figure 1B–D. https://cdn.elifesciences.org/articles/55800/elife-55800-fig1-data1-v2.xlsx Download elife-55800-fig1-data1-v2.xlsx Figure 1—source data 2 Source data for Figure 1E. https://cdn.elifesciences.org/articles/55800/elife-55800-fig1-data2-v2.pptx Download elife-55800-fig1-data2-v2.pptx Figure 1—source data 3 Source data for Figure 1F,G. https://cdn.elifesciences.org/articles/55800/elife-55800-fig1-data3-v2.xlsx Download elife-55800-fig1-data3-v2.xlsx Sepsis can lead to long-term functional deficits by immune cells (Jensen et al., 2018a). Therefore, to address whether the CLP-induced reduction in EAE disease was a durable phenomenon, we included a group of mice that had undergone CLP surgery 25 days prior to EAE immunization and evaluated the development of EAE disease (Figure 1a). Interestingly, even at this later time point CLP survivors continued to exhibit reduced disease severity (Figure 1b,c) and delayed disease onset (Figure 1d) relative to sham or no surgery controls, though disease severity was increased relative to day 5 CLP mice. The more severe disease relative to day 5 mice was due to a noted variability among the day 25 CLP mice, wherein some individuals developed severe disease and others had minimal disease. This difference in EAE severity suggests that the influence of the immunoparalysis state may wain with time, at least in some of the mice. Therefore, to capture the maximal state of suppression, we focused on comparisons of day five sham and CLP hosts. It is important to stress that sham surgery is the preferred control for assessing how the septic event influences the development of subsequent T-cell-mediated immune responses, since those mice experience the same surgical procedures as CLP mice (excluding those that initiate the septic event) (Cuenca et al., 2010; Sjaastad et al., 2020a). In particular, anesthesia, such as the ketamine used for the surgical procedures, can influence CD4 T cell function and capacity to induce EAE (Hou et al., 2016; Lee et al., 2017; Ohta et al., 2009). Thus, to ensure that differences observed were due to the septic event, rather than being imposed by surgery, we utilized sham surgery as a relevant control to CLP procedure for the remainder of our experiments. To further address how sepsis altered the systemic parameters of disease, we assessed serum cytokine/chemokine concentrations in sham and CLP hosts both prior (day 0) to and during EAE disease (day 15 post-EAE induction). Notably, the differences in clinical scores between sham and CLP hosts were associated with an altered pattern of expression by various systemic cytokines following EAE immunization (Figure 1—figure supplement 2a); this included numerous effector cytokines associated with EAE disease development (Figure 1—figure supplement 2b–i). Given the difference observed in disease between sham and CLP mice, in conjunction with an altered cytokine response, histopathologic scoring of the parenchymal and meningeal regions of the spinal cord during active disease was performed. Corresponding with their milder disease, CLP mice with EAE had less CNS pathology as highlighted by reduced inflammatory cell infiltrate (Figure 1e,H and E) in both the parenchyma (Figure 1f) and meninges (Figure 1g) of the spinal cord, the primary target of EAE inflammation. Further, CLP mice had milder axonal demyelination (Figure 1e, LFB) and vacuole formation, factors associated with EAE-induced paralysis. The difference in infiltrate observed extended to a reduced frequency and number of both microglia and infiltrating monocytes and macrophages present within the CNS in CLP hosts, relative to their sham counterparts (Figure 1—figure supplement 3a–e). A significant reduction in MHC II expression was also observed on both microglia and infiltrating monocytes/macrophages in CLP hosts compared to sham-treated mice (Figure 1—figure supplement 3f,h). This reduction compounded with the already reduced number of myeloid cells leading to ~100 fold reduction in MHC II-expressing cells in CLP hosts (Figure 1—figure supplement 3g,i). Prior studies have demonstrated alterations in MHC expression by myeloid cells which are dysregulated following sepsis (Jensen et al., 2020; Monneret et al., 2006; Siegler et al., 2018). MHC II expression is not only a marker of activation of these cell populations but also serves to present antigen to CD4 T cells, including autoreactive T cells that are critical mediators of disease in EAE. Therefore, this global infiltrate reduction in CLP hosts may reflect the lack of an autoimmune response in the CNS for which CD4 T cells are critical mediators. To address whether the reduction in histological inflammatory cell infiltrate reflected a difference in the accumulation of pathogenic antigen-specific CD4 T cells, total CNS (brain and spinal cord) was harvested from sham and CLP mice, following perfusion, at day 15 post-EAE induction (Figure 2a). Flow cytometric analysis of the CNS revealed a decreased frequency (Figure 2b,c) and number (Figure 2d) of antigen-experienced CD4+ CD11a+ cells in the CNS (Christiaansen et al., 2017). Further, tetramer staining of CD4 T cells reactive to the MOG antigen revealed a further reduction in the frequency (Figure 2e,f) and number (Figure 2g) of this pathogenic effector population. Figure 2 Download asset Open asset Fewer MOG-specific CD4 T cells are present in the CNS of CLP mice. (A) Experimental design: C57Bl/6 mice underwent either sham or CLP surgery. EAE induction occurred 5 days after surgery. Mice were perfused 15 days post-EAE induction and CNS was harvested. (B) Representative flow plots for CNS CD4 T cells, gated on lymphocytes, from sham and CLP mice. (C) Frequency and (D) number of CNS CD4 T cells in sham and CLP mice. (E) Representative flow plots for CNS MOG-specific CD4 T cells, gated on total CD4 T cells, from Naive (staining control), sham, and CLP mice. (F) Frequency and (G) number of CNS MOG-specific CD4 T cells in sham and CLP mice. Dashed line indicates staining and number from naive control mouse. Data are representative from two independent experiments with 8–10 mice per group. *p<0.05. Error bars represent the standard error of the mean. Figure 2—source data 1 Source data for Figure 2. https://cdn.elifesciences.org/articles/55800/elife-55800-fig2-data1-v2.xlsx Download elife-55800-fig2-data1-v2.xlsx These data suggest that the reduction in disease severity was potentially due to lack of autoantigen-specific CD4 T cells infiltrating the CNS; however, sepsis is also known to influence T cell function. Thus, direct ex vivo analysis of the MOG-specific CD4 T cells in the CNS was evaluated by intracellular cytokine staining at the same timepoint. No differences were observed in the frequencies of MOG-specific CD4 T cells producing pathogenic effector cytokines IFNγ, IL-17A, and TNFα without further stimulation directly ex vivo (Figure 3a,b; Figure 3—figure supplement 1; Lee et al., 2019). However, the numerical deficit in MOG-specific CD4 T cells led to a significant reduction in the number of IFNγ- and TNFα-producing cells with a trending decrease in IL-17A-producing cells (Figure 3c). This finding indicates the autoantigen-specific CD4 T cells present in the CNS after CLP surgery are functionally competent and potentially were the least impacted by the septic event. In summary, the data presented so far suggest that the numerical deficit in CNS-infiltrating pathogenic effectors might stem from insufficient priming and/or expansion of encephalitogenic CD4 T cells in the draining lymph node in the periphery. Figure 3 with 1 supplement see all Download asset Open asset Sepsis reduces the number of cytokine-producing MOG-specific CD4 T cells in the CNS. (A ) Representative flow plots of direct ex vivo IFNγ, IL-17A, and TNFα producing cells, gated on MOG-specific CD4 T cells, from sham (left) and CLP (right) mice. Numbers indicate frequency of cytokine-positive cells per quadrant. (B) Frequency and (C) number of IFNγ-, IL-17A-, and TNFα-producing MOG-specific CD4 T cells in the CNS of sham and CLP mice. Data are representative from two independent experiments with 8–10 mice per group. *p<0.05. Error bars represent the standard error of the mean. Figure 3—source data 1 Source data for Figure 3. https://cdn.elifesciences.org/articles/55800/elife-55800-fig3-data1-v2.xlsx Download elife-55800-fig3-data1-v2.xlsx Sepsis-induced loss of naive CD4 T cells is associated with a reduced number of autoantigen-specific CD4 T cell precursors To address whether the CLP hosts had a priming deficit for autoantigen-specific CD4 T cells, MOG-specific CD4 T cells were evaluated in the draining inguinal lymph node (iLN), 7 days after EAE induction (Figure 4a). This is a time during which CD4 T cells are being primed and expanding that precedes the development of clinical disease (Bischof et al., 2004). Similar to the CNS during peak disease, both the frequency (Figure 4b) and number (Figure 4c) of MOG-specific CD4 T cells were decreased in the iLN of CLP hosts. Figure 4 Download asset Open asset Sepsis reduces the number MOG-specific CD4 T cells present in sites of T cell priming following EAE induction. (A) Experimental design: C57Bl/6 mice underwent sham or CLP surgery. EAE induction occurred 5 days after surgery. Draining inguinal lymph nodes (iLN) were harvested 7 days after EAE disease induction. (B) Frequency and (C) number of MOG-specific CD4 T cells in the iLN of sham and CLP mice. Data are representative from three independent experiments with five mice per group. *p<0.05. Error bars represent the standard error of the mean. Figure 4—source data 1 Source data for Figure 4. https://cdn.elifesciences.org/articles/55800/elife-55800-fig4-data1-v2.xlsx Download elife-55800-fig4-data1-v2.xlsx Since lymphopenia is a hallmark of sepsis and differentially affects CD4 T cell precursor populations (Cabrera-Perez et al., 2015), a reduction in precursor numbers is one possibility for the decreased number of autoantigen-specific CD4 T cells in the iLN at the day 7 priming timepoint. Thus, the precursor frequency of MOG-specific naive CD4 T cells was interrogated through tetramer enrichment of splenic CD4 T cells 5 days after surgery (i.e. the day of EAE induction, Figure 5a). This analysis revealed a significant numerical loss of MOG-specific CD4 T cell precursors in CLP hosts. These data suggest CLP-induced lymphopenia may protect against the development of EAE by reducing the number of naive autoantigen-specific CD4 T cells. The subsequent loss of these naive cells would explain the corresponding reduction in the number of effector cells present in the both the lymph node and CNS following immunization. Figure 5 Download asset Open asset Sepsis reduces the number of naive MOG-specific CD4 T cell precursors. (A) Experimental design: C57Bl/6 mice underwent sham or CLP surgery. MOG-specific CD4 T cell precursors were enriched from the spleen 5 days after surgery via tetramer pulldown and enumerated. (B) Representative gating strategy for identifying MOG-specific CD4 T cell precursors from sham and CLP mice. Number of splenic MOG-specific CD4 T cell precursors in sham and CLP mice (B) 5 and (C) 25 days post-surgery. Frequency of antigen-experienced (CD44hi) (E) total and (F) MOG-specific CD4 T cells. Data are combined from two independent experiments with 8–13 mice per group. *p<0.05. Error bars represent the standard error of the mean. Figure 5—source data 1 Source data for Figure 5. https://cdn.elifesciences.org/articles/55800/elife-55800-fig5-data1-v2.xlsx Download elife-55800-fig5-data1-v2.xlsx To address whether the prior observation, that CLP hosts have a lasting decrease in the severity of EAE disease (Figure 1b–d), is also associated with a lasting numerical deficit, the number of MOG-specific CD4 T cells was enumerated in sham and CLP hosts 25 days post-surgery. Consistent with our observation of disparate disease development among CLP hosts, we observed a corresponding bifurcation in the number of MOG-specific CD4 T cells in individual mice (Figure 5d) further suggesting that the ability to initiate EAE disease might correspond to the number of MOG-specific CD4 T cells available. Additionally, similar to prior reports (Cabrera-Perez et al., 2016; Jensen et al., 2018a; Skirecki et al., 2020), an increased frequency of activated (CD44hi) CD4 T cells was observed in CLP hosts (Figure 5e). Importantly, this extended to an increase in the frequency of memory-like CD44hi cells among MOG-specific CD4 T cells in CLP hosts (Figure 5f). This increase in CD44 expression could occur either be through homeostatic proliferation following the severe lymphodepletion that occurs following a septic event or potentially through antigen release due to sepsis-induced tissue damage (Cabrera-Perez et al., 2016; Cabrera-Perez et al., 2015). Thus, even for those cells that do recover in numbers they are phenotypically and functionally distinct, which may further contribute to the differences in EAE disease development and progression following immunization. Loss of autoantigen-specific naive CD4 T cells is the determining factor in sepsis-induced impediment of EAE To further delineate the contributions of the sepsis influence directly on CD4 T cells or their environment, we utilized an adoptive transfer system wherein naive MOG-specific, TCR-transgenic 2D2 CD4 T cells were transferred into congenically distinct recipient mice. By having the fixed TCR of the 2D2 cells, the function of the 2D2 cells could be equally assessed between sham and CLP hosts. Mice receiving 2D2 cells 1 day prior to surgery (6 days prior to EAE induction) were part of the 'pre-transfer' group. Alternatively, a second cohort of mice received 2D2 cells 4 days post-surgery (1 day prior to EAE induction), a time at which sepsis-associated inflammation has resolved and the immunoparalysis state is established (Danahy et al., 2019b), were in the 'post-transfer' group. 2D2 cells in the pre-transfer group are exposed to both the intrinsic and extrinsic changes that sepsis induces. In contrast, the 2D2 cells in the post-transfer experiments were only influenced by the sepsis-induced changes in the environment, such as reduced DC function (Strother et al., 2016), reduced capacity to traffic across endothelium (Danahy et al., 2017), and alterations in the proportion of regulatory CD4 and CD8 T cells (Sharma et al., 2015; Sinha et al., 2015). These impairments are all likely to be present in the context of post-septic environment and may influence the development of EAE. Thus, the post-septic environmental factors which influence CD4 T cells can be assessed by transferring cells into the host after the immunoparalysis state has been established. Further, by comparing the pre- and post-transfer cells from sham and CLP mice the intrinsic and extrinsic influences of sepsis can be parsed. Thus, to address the intrinsic and extrinsic impact of sepsis on priming of CD4 T cells, 2D2 cell number, proliferation, as assessed by recent proliferation marker Ki67 (Miller et al., 2018), and apoptosis, as assessed by presence of active caspase 3/7 (FLICA+) and membrane depolarization (PI+), were evaluated in the iLN 7 days post-EAE induction (Figure 6a,b). Notably, cells that were transferred prior to surgery were numerically diminished in CLP hosts relative to sham counterparts (Figure 6c) recapitulating the observations with endogenous MOG-specific CD4 T cells (see Figure 5). Strikingly, 2D2 cells that were transferred after surgery were numerically equivalent between sham and CLP hosts, suggesting the septic environment (e.g. diminished DC number/function, increased proportion of regulatory CD8 and/or CD4 T cells) did not limit their capacity to expand (Figure 6c). Further, the transferred 2D2 cells from all groups had a similar proportion of recently proliferated as well as apoptotic (FLICA+ PI+) cells (Figure 6d,e). Given that the post-transfer groups had equivalent cell numbers, proliferation, and apoptosis; these data demonstrate that autoantigen CD4 T cells are not limited by the post-septic environment in their capacity to be primed and expand. Thus, while sepsis notably induces a variety of T-cell-extrinsic immunologic impairments (Danahy et al., 2017; Sharma et al., 2015; Sinha et al., 2015; Strother et al., 2016), these impairments can be overcome in the context of EAE immunization. In addition, there was no observed difference in the expression of several canonical extrinsic death-inducing proteins known to limit the expansion of CD4 T cells (i.e. Fas, FasL, TRAIL) between sham and CLP hosts for either the pre- or post-transfer groups (Figure 6—figure supplement 1) further supporting the notion that survival differences do not account for the numerical deficit in CD4 T cells exposed to sepsis. Therefore, pre-transfer 2D2 cells proliferate and die equivalently in both sham and CLP hosts, indicating that sepsis does not intrinsically impair the capacity of these cells to proliferate in response to cognate antigen recognition. Figure 6 with 2 supplements see all Download asset Open asset Sepsis reduces the number of MOG-specific CD4 T cells but not their capacity to proliferate. (A) Experimental design: Thy1.2 C57Bl/6 mice were separated into Pre- and Post-transfer groups. The Pre-transfer group received 5 × 103 naive Thy1.1 2D2 TCR-Tg CD4 T cells 1 day before sham or CLP surgery. The Post-transfer group underwent sham or CLP surgery then received 5 × 103 naive Thy1.1 2D2 TCR-Tg CD4 T cells 4 days later. EAE was induced in both the Pre- and Post-transfer groups 5 days after surgery (Pre: 6 days post 2D2 T cell transfer; Post: 1 day post 2D2 T cell transfer). iLN were harvested 7 days after the transfer. (B) Representative gating strategy for identifying transferred 2D2 TCR-Tg CD4 T cells, their expression of the proliferation marker Ki67, and markers of apoptosis (activated caspase3/7 with propidium iodide) from

The current SARS-CoV-2 pandemic is accompanied by high morbidity and mortality rates, and there is a compelling need for effective vaccines and therapeutic agents to lessen the severity of COVID-19 disease. Appropriate animal models are essential for testing of vaccines and therapeutics and for mechanistic studies of infection and the host response. The Spike (S) protein of SARS-COV-2 has a high affinity for the human ACE2 receptor, which is expressed on multiple cell types including alveolar epithelial and vascular endothelial cells. Wild-type mice are not susceptible to developing coronavirus-mediated diseases. Accordingly, several human (h)ACE2 transgenic mouse models have been developed for coronavirus research. However, these mice have failed to closely mimic important aspects of the human immunopathological responses to SARS-CoV-2. We report herein that DRAGA (HLA-A2.HLA-DR4.Rag1KO.IL-2Rγc KO.NOD) mice infused with human hematopoietic stem cells from cord blood reconstitute a fully functional human immune system, as well as engraft human epithelial and endothelial cells, sustain SARS-CoV-2 infection, and develop severe COVID-19-like symptoms. In pilot experiments, infected mice developed parenchymal and epithelial lung infiltrations with granzyme B+ and perforin+ CD8+ T cells and alveolar CD61+ microthrombi, mimicking human immunopathological responses to SARS-CoV-2. We propose the DRAGA mouse as a novel pre-clinical tool for studying COVID-19 immunopathology and human immune responses to SARS-CoV-2, including events leading to the cytokine storm and coagulopathies, as well as for testing of candidate vaccines and therapeutics.

BACKGROUND:Human-immune-system humanized mouse models can bridge the gap between humans and conventional mice for testing human vaccines. The HLA-expressing humanized DRAGA (HLA-A2.HLA-DR4.Rag1KO.IL2RγcKO.NOD) mice reconstitute a functional human-immune-system and sustain the complete life cycle of Plasmodium falciparum. Herein, the DRAGA mice were investigated for immune responses following immunization with live P. falciparum sporozoites under chloroquine chemoprophylaxis (CPS-CQ), an immunization approach that showed in human trials to confer pre-erythrocytic immunity.RESULTS:The CPS-CQ immunized DRAGA mice (i) elicited human CD4 and CD8 T cell responses to antigens expressed by P. falciparum sporozoites (Pfspz) and by the infected-red blood cells (iRBC). The Pfspz-specific human T cell responses were found to be systemic (spleen and liver), whereas the iRBCs-specific human T cell responses were more localized to the liver, (ii) elicited stronger antibody responses to the Pfspz than to the iRBCs, and (iii) they were protected against challenge with infectious Pfspz but not against challenge with iRBCs.CONCLUSIONS:The DRAGA mice represent a new pre-clinical model to investigate the immunogenicity and protective efficacy of P. falciparum malaria vaccine candidates.

The mucosal tissues of the gut and female reproductive tract (FRT) are susceptible to pathogen infections including bacteria, viruses, and parasites, and are also the targets for immune disorders such as Crohn's disease, inflammatory bowel disease (IBD), and many types of cancers. However, the role of the mucosal immune cells to control these diseases is largely unknown. The limited availability of human mucosal biopsy tissue and the low number of cells that can be isolated from these tissues hampers the characterization of the phenotype and function of human mucosal immune cell subsets. Therefore, human-immune-system humanized mice are surrogate models to investigate the human mucosal immune cell responses during the course of the disease. The current protocols used to harvest the immune cells from the mucosal tissues, however, result in low recovery of cells with poor viability. We have established a novel protocol, which results in a high yield of human lymphocytes with high viability to overcome this issue. The immune cells obtained from a single DRAG mouse by our protocol were sufficient for conducting functional assays and for flow cytometry analyses including phenotypic, exhaustion, and functional panels.

Pandemic outbreaks of influenza type A viruses have resulted in numerous fatalities around the globe. Since the conventional influenza vaccines (CIV) provide less than 20% protection for individuals with weak immune system, it has been considered that broadly cross-neutralizing antibodies may provide a better protection. Herein, we showed that a recently generated humanized mouse (DRAGA mouse; HLA-A2. HLA-DR4. Rag1KO. IL-2Rgc KO. NOD) that lacks the murine immune system and expresses a functional human immune system can be used to generate cross-reactive, human anti-influenza monoclonal antibodies (hu-mAb). DRAGA mouse was also found to be suitable for influenza virus infection, as it can clear a sub-lethal infection and sustain a lethal infection with PR8/A/34 influenza virus. The hu-mAbs were designed for targeting a human B-cell epitope (180WGIHHPPNSKEQ QNLY195) of hemagglutinin (HA) envelope protein of PR8/A/34 (H1N1) virus with high homology among seven influenza type A viruses. A single administration of HA180-195 specific hu-mAb in PR8-infected DRAGA mice significantly delayed the lethality by reducing the lung damage. The results demonstrated that DRAGA mouse is a suitable tool to (i) generate heterotype cross-reactive, anti-influenza human monoclonal antibodies, (ii) serve as a humanized mouse model for influenza infection, and (iii) assess the efficacy of anti-influenza antibody-based therapeutics for human use.

It is with great regret and sadness that we announce the passing of the founding Editor-in-Chief of International Reviews of Immunology, Constantin A. Bona, M.D., Ph.D., Professor Emeritus of Micro...

Background. Plasmodium yoelii 17XNL is a nonlethal malaria strain in mice of different genetic backgrounds including the C57BL/6 mice (I-Ab/I-Enull) used in this study as a control strain. We have compared the trends of blood stage infection with the nonlethal murine strain of P. yoelii 17XNL malaria protozoan in immunocompetent Nonobese Diabetic (NOD) mice prone to type 1 diabetes (T1D) and C57BL/6 mice (control mice) that are not prone to T1D and self-cure the P. yoelii 17XNL infection. Prediabetic NOD mice could not mount a protective antibody response to the P. yoelii 17XNL-infected red blood cells (iRBCs), and they all succumbed shortly after infection. Our data suggest that the lack of anti-P. yoelii 17XNL-iRBCs protective antibodies in NOD mice is a result of parasite-induced, Foxp3+ T regulatory (Treg) cells able to suppress the parasite-specific antibody secretion. Conclusions. The NOD mouse model may help in identifying new mechanisms of B-cell evasion by malaria parasites. It may also serve as a more accurate tool for testing antimalaria therapeutics due to the lack of interference with a preexistent self-curing mechanism present in other mouse strains.

Humanized mice expressing Human Leukocyte Antigen (HLA) class I or II transgenes have been generated, but the role of class I vs class II on human T and B cell reconstitution and function has not been investigated in detail. Herein we show that NRG (NOD.RagKO.IL2RγcKO) mice expressing HLA-DR4 molecules (DRAG mice) and those co-expressing HLA-DR4 and HLA-A2 molecules (DRAGA mice) did not differ in their ability to develop human T and B cells, to reconstitute cytokine-secreting CD4 T and CD8 T cells, or to undergo immunoglobulin class switching. In contrast, NRG mice expressing only HLA-A2 molecules (A2 mice) reconstituted lower numbers of CD4 T cells but similar numbers of CD8 T cells. The T cells from A2 mice were deficient at secreting cytokines, and their B cells could not undergo immunoglobulin class switching. The inability of A2 mice to undergo immunoglobulin class switching is due to deficient CD4 helper T cell function. Upon immunization, the frequency and cytotoxicity of antigen-specific CD8 T cells in DRAGA mice was significantly higher than in A2 mice. The results indicated a multifactorial effect of the HLA-DR4 transgene on development and function of human CD4 T cells, antigen-specific human CD8 T cells, and immunoglobulin class switching.

The number of humanized mouse models for the human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and other infectious diseases has expanded rapidly over the past 8 years. Highly immunodeficient mouse strains, such as NOD/SCID/gamma chain(null) (NSG, NOG), support better human hematopoietic cell engraftment. Another improvement is the derivation of highly immunodeficient mice, transgenic with human leukocyte antigens (HLAs) and cytokines that supported development of HLA-restricted human T cells and heightened human myeloid cell engraftment. Humanized mice are also used to study the HIV reservoir using new imaging techniques. Despite these advances, there are still limitations in HIV immune responses and deficits in lymphoid structures in these models in addition to xenogeneic graft-versus-host responses. To understand and disseminate the improvements and limitations of humanized mouse models to the scientific community, the NIH sponsored and convened a meeting on April 15, 2015 to discuss the state of knowledge concerning these questions and best practices for selecting a humanized mouse model for a particular scientific investigation. This report summarizes the findings of the NIH meeting.

Several human HLA alleles have been found associated with type 1 diabetes (T1D), but their precise role is not clearly defined. Herein, we report that a human MHC class II (HLA-DR*0401) allele transgene that has been expressed into NOD (H-2(g7)I-E(null)) mice prone to T1D rendered the mice resistant to the disease. T1D resistance occurred in the context of multi-point T-cell alterations such as: (i) skewed CD4/CD8 T-cell ratio, (ii) decreased size of CD4(+)CD44(high) T memory pool, (iii) aberrant TCR Vβ repertoire, (iv) increased neonatal number of Foxp3(+) and TR-1(+) regulatory cells, and (v) reduced IFN-γ inflammatory response vs. enhanced IL-10 suppressogenic response of T-cells upon polyclonal and antigen-specific stimulation. The T-cells from NOD/DR4 Tg mice were unable to induce or suppress diabetes in NOD/RAG deficient mice. This study describes a multifaceted regulatory function of the HLA-DR*0401 allele strongly associated with the lack of T1D development in NOD mice.

CD4 + T follicular helper cells (T FH ) in germinal centers are required for maturation of B-cells. While the role of T FH -cells has been studied in blood and lymph nodes of HIV-1 infected individuals, its role in the mucosal tissues has not been investigated. We show that the gut and female reproductive tract (FRT) of humanized DRAG mice have a high level of human lymphocytes and a high frequency of T FH (CXCR5 + PD-1 ++ ) and precursor-T FH (CXCR5 + PD-1 + ) cells. The majority of T FH -cells expressed CCR5 and CXCR3 and are the most permissive to HIV-1 infection. A single low-dose intravaginal HIV-1 challenge of humanized DRAG mice results in 100% infectivity with accumulation of T FH -cells mainly in the Peyer’s patches and FRT. The novel finding of T FH -cells in the FRT may contribute to the high susceptibility of DRAG mice to HIV-1 infection. This mouse model thus provides new opportunities to study T FH -cells and to evaluate HIV-1 vaccines.

Perkinsus marinus (Phylum Perkinsozoa) is a marine protozoan parasite responsible for "Dermo" disease in oysters, which has caused extensive damage to the shellfish industry and estuarine environment. The infection prevalence has been estimated in some areas to be as high as 100%, often causing death of infected oysters within 1-2 years post-infection. Human consumption of the parasites via infected oysters is thus likely to occur, but to our knowledge the effect of oral consumption of P. marinus has not been investigated in humans or other mammals. To address the question we used humanized mice expressing HLA-DR4 molecules and lacking expression of mouse MHC-class II molecules (DR4.EA(0)) in such a way that CD4 T cell responses are solely restricted by the human HLA-DR4 molecule. The DR4.EA(0) mice did not develop diarrhea or any detectable pathology in the gastrointestinal tract or lungs following single or repeated feedings with live P. marinus parasites. Furthermore, lymphocyte populations in the gut associated lymphoid tissue and spleen were unaltered in the parasite-fed mice ruling out local or systemic inflammation. Notably, naïve DR4.EA(0) mice had antibodies (IgM and IgG) reacting against P. marinus parasites whereas parasite specific T cell responses were undetectable. Feeding with P. marinus boosted the antibody responses and stimulated specific cellular (IFNγ) immunity to the oyster parasite. Our data indicate the ability of P. marinus parasites to induce systemic immunity in DR4.EA(0) mice without causing noticeable pathology, and support rationale grounds for using genetically engineered P. marinus as a new oral vaccine platform to induce systemic immunity against infectious agents.