Search

Introduction Classically IgA in the gut prevents the invasion of microorganisms to systemic organs through the process of neutralization and immune exclusion. Interestingly, recent reports suggest that IgA might help in biofilm formation and promote bacterial growth inside the intestine. Methods In this study, we used flow cytometry, ELISA and chemical models of colitis to test whether the quality and quantity of IgA can select for bacterial persistence in the gut. Results We found that members of Proteobacteria such as γ-Proteobacteria and SFB are preferentially coated by IgA in WT mice. In partial absence of either T-dependent or T-independent IgA responses, there are no significant differences in the frequency of bacteria coated with IgA in mice. However, in, Rag-/- mice that lack all antibodies, had a severe reduction in Proteobacteria and were resistant to DSS induced colitis, suggesting that secretory IgA might be essential for differential retention of these taxa in the mouse gut. Rag-/- littermates in the F2 generation generated from (B6 x Rag-/-) F1 mice acquired the underrepresented bacteria taxa such as γ-Proteobacteria through vertical transmission of flora and died soon after weaning possibly due to the acquired flora. Additionally, continued exposure of Rag-/- mice to B6 flora by cohousing mice led to the acquisition of γ-Proteobacteria and to mortality. Conclusion Together, our results indicate that host survival in the complete absence of an IgA response necessitates the exclusion of certain bacterial taxa from the gut microbiome.

Protein synthesis is tightly regulated by both gene-specific and global mechanisms to match the metabolic and proliferative demands of the cell. While the regulation of global protein synthesis in response to mitogen or stress signals is relatively well understood in multiple experimental systems, how different cell types fine-tune their basal protein synthesis rate is not known. In a previous study, we showed that resting B and T lymphocytes exhibit dramatic differences in their metabolic profile, with implications for their post-activation function. Here, we show that resting B cells, despite being quiescent, exhibit increased protein synthesis in vivo as well as ex vivo. The increased protein synthesis in B cells is driven by mTORC1, which exhibits an intermediate level of activation in these cells when compared with resting T cells and activated B cells. A comparative analysis of the transcriptome and translatome of these cells indicates that the genes encoding the MHC Class II molecules and their chaperone CD74 are highly translated in B cells. These data suggest that the translatome of B cells shows enrichment for genes associated with antigen processing and presentation. Even though the B cells exhibit higher mTORC1 levels, they prevent the translational activation of TOP mRNAs, which are mostly constituted by ribosomal proteins and other translation factors, by upregulating 4EBP1 levels. This mechanism may keep the protein synthesis machinery under check while enabling higher levels of translation in B cells.

Studies with gene‐deficient and gnotobiotic mice have identified many host and microbial factors that contribute to induced colitis, but information on whether specific factors determine susceptibility under more physiological conditions is lacking. Using wild‐type strains that differ in their IgA response but harbor a diverse gut microbiome, we found that the IgA‐high strain CBA/CaJ (CBA) is resistant to acute colitis induced with dextran sodium sulfate (DSS), unlike the IgA‐low strain C57BL/6 (B6). Resistance was associated with extensive IgA‐coating of fecal bacteria, lower fecal bacterial loads and greater abundance of barrier‐protective transcripts in colonic tissues under homeostatic conditions. Fecal microbial transplant (FT) experiments revealed that disease induction in B6 mice was associated with a cohort of bacteria that are not targeted by IgA. However, CBA mice continued to be resistant to colitis induction following FTs from B6 mice, indicating that they are able to contain such colitogenic members. In support of a role for bacterial exclusion in resistance, oral administration of immunoglobulins decreased DSS‐induced disease in B6 mice. In F(1) mice derived separately with CBA and B6 dams and in F(1) mice backcrossed to the two parental strains, resistance segregated with the IgA response of the pups and not with barrier‐associated transcripts or bacterial loads. Interestingly, B6 pups foster‐nursed on CBA dams continued to be susceptible in later life, whereas CBA pups foster‐nursed on B6 dams continued to be resistant. Together, the data indicate that a high‐IgA response in adult life can protect against colitis and compensate for IgA deficiency in early life.

We have previously demonstrated co‐receptor level‐associated functional heterogeneity in apparently homogeneous naive peripheral CD4 T cells, dependent on MHC‐mediated tonic signals. Maturation pathways can differ between naive CD4 and naive CD8 cells, so we tested whether the latter showed similar co‐receptor level‐associated functional heterogeneity. We report that, when either polyclonal and T‐cell receptor (TCR)‐transgenic monoclonal peripheral naive CD8 T cells from young mice were separated into CD8(hi) and CD8(lo) subsets, CD8(lo) cells responded poorly, but CD8(hi) and CD8(lo) subsets of CD8 single‐positive (SP) thymocytes responded similarly. CD8(lo) naive CD8 T cells were smaller and showed lower levels of some cell‐surface molecules, but higher levels of the negative regulator CD5. In addition to the expected peripheral decline in CD8 levels on transferred naive CD8 T cells in wild‐type (WT) but not in MHC class I‐deficient recipient mice, short‐duration naive T‐cell–dendritic cell (DC) co‐cultures in vitro also caused co‐receptor down‐modulation in CD8 T cells but not in CD4 T cells. Constitutive pZAP70/pSyk and pERK levels ex vivo were lower in CD8(lo) naive CD8 T cells and dual‐specific phosphatase inhibition partially rescued their hypo‐responsiveness. Bulk mRNA sequencing showed major differences in the transcriptional landscapes of CD8(hi) and CD8(lo) naive CD8 T cells. CD8(hi) naive CD8 T cells showed enrichment of genes involved in positive regulation of cell cycle and survival. Our data show that naive CD8 T cells show major differences in their signaling, transcriptional and functional landscapes associated with subtly altered CD8 levels, consistent with the possibility of peripheral cellular aging.

ObjectivesTo assess seroprevalence of anti-SARS-CoV-2 antibodies in a densely populated urban Indian settings and its implications for disease trends and protective immunity.DesignCross-sectional sero-epidemiological survey linked with administrative reporting of COVID-19 testing data.SettingsPune city in western IndiaMain outcome measurePrevalence of anti-SARS-CoV-2 spike protein antibodies were estimated and along with correlates of virus neutralisation and other immune and inflammatory markers.ResultsSeropositivity was extensive (51·3%; 95%CI 39·9 to 62·4) but varied widely in the five localities tested, ranging from 35·8% to 66·4%. Seropositivity was higher in crowded living conditions in the slums (OR 1·91), and was lower in those 65 years or older (OR 0·59). The infection-fatality ratio was estimated at 0.21%. Post survey, COVID-19 incidence was lower in areas noted to have higher seroprevalence. Substantial virus-neutralising activity was observed in seropositive individuals, but with considerable heterogeneity in the immune response and possible age-dependent diversity in the antibody repertoire.ConclusionDespite crowded living conditions having facilitated widespread transmission, the variability in seroprevalence in localities that are in geographical proximity indicates a heterogenous spread of infection. Declining infection rates in areas with high seropositivity suggest population-level protection. It is also supported by substantial neutralising activity in asymptomatically infected individuals. This is the first report of a significantly high proportion of protective immune response among asymptomatic individuals in the population. The heterogeneity in antibody levels and neutralisation capacity indicates the existence of immunological sub-groups of functional interest.Trial registrationRegistered with the Clinical Trials Registry of India (CTRI/2020/07/026509)

ProblemWhile the testes represent an immune privileged organ, there is evidence that systemic inflammation is accompanied by local inflammatory responses. We therefore examined if transient systemic inflammation caused any inflammatory and functional consequences in murine testes.Method of StudyUsing a single systemic administration of Toll-like receptor (TLR) agonists [lipopolysaccharide (LPS) or peptidoglycan (PG) or polyinosinic-polycytidylic acid (polyIC)] in young adult male mice, we assessed testicular immune-inflammatory landscape and reproductive functionality.ResultsOur findings demonstrated a significant induction of testicular TNF-α, IL-1β and IL-6 transcripts within 24 h of TLR agonist injection. By day 6 these cytokine levels returned to baseline. While there was no change in caudal sperm counts at early time points, eight weeks later, two-fold decrease of sperm count and reduced testicular testosterone levels was evident. When these mice were subjected to mating studies, no differences in mating efficiencies or litter sizes were observed compared to controls. Nonetheless, the neonatal weights of progeny from LPS/PG/polyIC treated sires were significantly lower than controls. Postnatal weight gain up to three weeks was also slower in the progeny of LPS/polyIC treated sires. Placental weights at 17.5 days post-coitum were significantly lower in females mated to LPS and polyIC treated males. Given this likelihood of an epigenetic effect, we found lower testicular levels of histone methyl-transferase enzyme, mixed-lineage leukemia-1, in mice given LPS/PG/polyIC eight weeks earlier.ConclusionExposure to transient systemic inflammation leads to transient local inflammation in the testes, with persistent sperm-mediated consequences for fetal development.

Fever is a conserved and prominent response to infection. Yet, the issue of how CD4 T cell responses are modulated if they occur at fever temperatures remains poorly addressed. We have examined the priming of naive CD4 T cells in vitro at fever temperatures, and we report notable fever-mediated modulation of their cytokine commitment. When naive CD4 T cells were primed by plate-bound anti-CD3 and anti-CD28 monoclonal antibodies at moderate fever temperature (39 °C), they enhanced commitment to IL4/5/13 (Th2) and away from IFNg (Th1). This was accompanied by up-regulation of the Th2-relevant transcription factor GATA3 and reduction in the Th1-relevant transcription factor Tbet. Fever sensing by CD4 T cells involved transient receptor potential vanilloid cation channels (TRPVs) since TRPV1/TRPV4 antagonism blocked the febrile Th2 switch, while TRPV1 agonists mediated a Th2 switch at 37 °C. The febrile Th2 switch was IL4 independent, but a γ-secretase inhibitor abrogated it, and it was not found in Notch1-null CD4 T cells, identifying the Notch pathway as a major mediator. However, when naive CD4 T cells were primed via antigen and dendritic cells (DCs) at fever temperatures, the Th2 switch was abrogated via increased production of IL12 from DCs at fever temperatures. Thus, immune cells directly sense fever temperatures with likely complex physiological consequences.

We previously reported that Indian paediatric patients with atypical haemolytic–uraemic syndrome (aHUS) showed high frequencies of anti‐complement factor H (FH) autoantibodies that are correlated with homozygous deletion of the genes for FH‐related proteins 1 and 3 (FHR1 and FHR3) (FHR1/3 (–/–)). We now report that Indian paediatric aHUS patients without anti‐FH autoantibodies also showed modestly higher frequencies of the FHR1/3 (–/–) genotype. Further, when we characterized epitope specificities and binding avidities of anti‐FH autoantibodies in aHUS patients, most anti‐FH autoantibodies were directed towards the FH cell‐surface anchoring polyanionic binding site‐containing C‐terminal short conservative regions (SCRs) 17–20 with higher binding avidities than for native FH. FH SCR17–20‐binding anti‐FH autoantibodies also bound the other cell‐surface anchoring polyanionic binding site‐containing region FH SCR5–8, at lower binding avidities. Anti‐FH autoantibody avidities correlated with antibody titres. These anti‐FH autoantibody characteristics did not differ between aHUS patients with or without the FHR1/3 (–/–) genotype. Our data suggest a complex matrix of interactions between FHR1‐FHR3 deletion, immunomodulation and anti‐FH autoantibodies in the aetiopathogenesis of aHUS.

T cell response magnitudes increase with increasing antigenic dosage. However, it is unclear whether ligand density only modulates the proportions of responding ligand-specific T cells or also alters responses at the single cell level. Using brief (3 h) exposure of TCR-transgenic mouse CD8 T cells in vitro to varying densities of cognate peptide-MHC ligand followed by ligand-free culture in IL-2, we found that ligand density determined the frequencies of responding cells but not the expression levels of the early activation marker molecule, CD69. Cells with low glucose uptake capacity and low protein synthesis rates were less ligand-sensitive, implicating metabolic competence in the response heterogeneity of CD8 T cell populations. Although most responding cells proliferated, ligand density was associated with time of entry into proliferation and with the extent of cell surface TCR downmodulation. TCR internalization was associated, regardless of the ligand density, with rapidity of c-myc induction, loss of the cell cycle inhibitor p27kip1, metabolic reprogramming, and cell cycle entry. A low affinity peptide ligand behaved, regardless of ligand density, like a low density, high affinity ligand in all these parameters. Inhibition of signaling after ligand exposure selectively delayed proliferation in cells with internalized TCRs. Finally, internalized TCRs continued to signal and genetic modification of TCR internalization and trafficking altered the duration of signaling in a T cell hybridoma. Together, our findings indicate that heterogeneity among responding CD8 T cell populations in their ability to respond to TCR-mediated stimulation and internalize TCRs mediates detection of ligand density or affinity, contributing to graded response magnitudes.

Activated T-cells make both interleukin-2 (IL2) and its high-affinity receptor component CD25. Regulatory CD4 T-cells (Treg cells) do not make IL2, and the IL2-CD25 circuit is considered a paracrine circuit crucial in their generation and maintenance. Yet, all T-cells are capable of making IL2 at some stage during differentiation, making a cell-intrinsic autocrine circuit additionally possible. When we re-visited experiments with mixed bone marrow chimeras using a wide range of ratios of wild-type (WT) and IL2-/- genotype progenitors, we found that, as expected, thymic Treg cells were almost equivalent between WT and IL2-/- genotypes at ratios with WT prominence. However, at WT-limiting ratios, the IL2-/- genotype showed lower thymic Treg frequencies, indicating a role for cell-intrinsic autocrine IL2 in thymic Treg generation under IL2-limiting conditions. Further, peripheral IL2-/- naive CD4 T-cells showed poor conversion to inducible Tregs (pTregs) both in vivo and in vitro, again indicating a significant role for cell-intrinsic autocrine IL2 in their generation. Peripherally, the IL2-/- genotype was less prominent at all WT:IL2-/- ratios among both thymic Tregs (tTregs) and pTregs, adoptively transferred IL2-/- Tregs showed poorer survival than WT Tregs did, and RNA-seq analysis of WT and IL2-/- Tregs showed interesting differences in the T-cell receptor and transforming growth factor-beta-bone morphogenetic protein-JNK pathways between them, suggesting a non-titrating role for cell-intrinsic autocrine IL2 in Treg programming. These data indicate that cell-intrinsic autocrine IL2 plays significant roles in Treg generation and maintenance.

The immunological roles of the nuclear factor-kappaB (NF-κB) pathway are mediated via the canonical components in immune responses and via non-canonical components in immune organogenesis and homeostasis, although the two components are capable of crosstalk. Regulatory CD4 T cells (Tregs) are homeostatically functional and represent an interesting potential meeting point of these two NF-κB components. We show that mice deficient in the non-canonical NF-κB component gene Nfkb2 (p100) had normal thymic development and suppressive function of Tregs. However, they had enhanced frequencies of peripheral ‘effector-phenotype’ Tregs (eTregs). In bi-parental chimeras of wild-type (WT) and Nfkb2−/− mice, the Nfkb2−/− genotype was over-represented in Tregs, with a further increase in the relative prominence of eTregs. Consistent with distinct properties of eTregs, the Nfkb2−/− genotype was more prominent in Tregs in extra-lymphoid tissues such as liver in the bi-parental chimeras. The Nfkb2−/− Tregs also displayed greater survival, activation and proliferation in vivo. These Nfkb2−/− Tregs showed higher nuclear NF-κB activity mainly comprising of RelB-containing dimers, in contrast to the prominence of cRel- and RelA-containing dimers in WT Tregs. Since p100 is an inhibitor of RelB activation as well as a participant as cleaved p52 in RelB nuclear activity, we tested bi-parental chimeras of WT and Relb−/− mice, and found normal frequencies of Relb−/− Tregs and eTregs in these chimeric mice. Our findings confirm and extend recent data, and indicate that p100 normally restrains RelB-mediated Treg activation, and in the absence of p100, p50-RelB dimers can contribute to Treg activation.

Immune parameters show characteristic normal baseline levels and variance in the population. We characterised the degree of inter-individual and within-individual variation over one-year time period in 33 immune cell subsets by flow cytometry in peripheral blood mononuclear cells from 43 healthy young adult volunteers. Our analysis revealed that immune subsets that showed low variability between individuals also showed low short-term fluctuations within-individuals, as well as concordance in siblings. However, when baseline levels and degree of fluctuation were considered together, individuals failed to cluster into discreet groups. Together, the data reveal complex inter-relationships between immune subsets in individuals, and provide insights into the observed heterogeneity between individuals and between multiple immune subsets.

Activation of B and T lymphocytes leads to major remodelling of the metabolic landscape of the cells enabling their post-activation functions. However, naive B and T lymphocytes also show metabolic differences, and the genesis, nature and functional significance of these differences are not yet well understood. Here we show that resting B-cells appeared to have lower energy demands than resting T-cells as they consumed lower levels of glucose and fatty acids and produced less ATP. Resting B-cells are more dependent on OXPHOS, while T-cells show more dependence on aerobic glycolysis. However, despite an apparently higher energy demand, T lineage cells showed lower rates of protein synthesis than equivalent B lineage stages. These metabolic differences between the two lineages were established early during lineage differentiation, and were functionally significant. Higher levels of protein synthesis in B-cells were associated with increased synthesis of MHC class II molecules and other proteins associated with antigen internalization, transport and presentation. The combination of higher energy demand and lower protein synthesis in T-cells was consistent with their higher ATP-dependent motility. Our data provide an integrated perspective of the metabolic differences and their functional implications between the B and T lymphocyte lineages.

A homozygous 83-kb deletion encompassing the genes for complement factor-H-related proteins 1 and 3 (FHR 1, FHR3) is known as a risk factor for some immune inflammatory disorders. However, the functional relevance of this FHR1/3 deletion is relatively unexplored. Globally, healthy populations of all ethnic groups tested show an 8-10% prevalence of homozygosity for this deletion polymorphism. We have begun to compare the peripheral leucocyte phenotype and functionality between FHR1/3-/- and FHR1/3+/+ healthy adult individuals. We report that the two groups show significant differences in their peripheral blood innate leucocyte subset composition, although the adaptive immune subsets are similar between them. Specifically, FHR1/3-/- individuals show higher frequencies of patrolling monocytes and lower frequencies of classical monocytes than FHR1/3+/+ individuals. Similarly, FHR1/3-/- individuals show higher frequencies of plasmacytoid dendritic cells (pDCs) and lower frequencies of myeloid DCs (mDCs) than FHR1/3+/+ individuals. Notably, classical monocytes specifically showed cell-surface-associated factor H (FH), and cells from the FHR1/3-/- group had somewhat higher surface-associated FH levels than those from FHR1/3+/+ individuals. FHR1/3-/- monocytes also showed elevated secretion of TNF-α, IL-1β, and IL-10 in response to TLR7/8 or TLR4 ligands. Similarly, FHR1/3-/- mDCs and pDCs showed modest but evident hyper-responsiveness to TLR ligands. Our findings, that the FHR1/3-/- genotype is associated with significant alterations of both the relative prominence and the functioning of monocyte and DC subsets, may be relevant in understanding the mechanism underlying the association of the genotype with immune inflammatory disorders.

Memory T and B lymphocyte numbers are thought to be regulated by recent and cumulative microbial exposures. We report here that memory-phenotype lymphocyte frequencies in B, CD4 and CD8 T-cells in 3-monthly serial bleeds from healthy young adult humans were relatively stable over a 1-year period, while recently activated -B and -CD4 T cell frequencies were not, suggesting that recent environmental exposures affected steady state levels of recently activated but not of memory lymphocyte subsets. Frequencies of memory B and CD4 T cells were not correlated, suggesting that variation in them was unlikely to be determined by cumulative antigenic exposures. Immunophenotyping of adult siblings showed high concordance in memory, but not of recently activated lymphocyte subsets, suggesting genetic regulation of memory lymphocyte frequencies. To explore this possibility further, we screened effector memory (EM)-phenotype T cell frequencies in common independent inbred mice strains. Using two pairs from these strains that differed predominantly in either CD4EM and/or CD8EM frequencies, we constructed bi-parental bone marrow chimeras in F1 recipient mice, and found that memory T cell frequencies in recipient mice were determined by donor genotypes. Together, these data suggest cell-autonomous determination of memory T niche size, and suggest mechanisms maintaining immune variability.

Sumi Krishna and Gita Chadha (Eds), Feminists and Science: Critiques and Changing Perspectives in India, Vol. 1. Kolkata: Stree, 2015. 348 pages. ₹ 500, ISBN 978-93-813-4507-8.

Lymphotoxin‐beta receptor (LTβR) present on stromal cells engages the noncanonical NF‐κB pathway to mediate RelB‐dependent expressions of homeostatic chemokines, which direct steady‐state ingress of naive lymphocytes to secondary lymphoid organs (SLOs). In this pathway, NIK promotes partial proteolysis of p100 into p52 that induces nuclear translocation of the RelB NF‐κB heterodimers. Microbial infections often deplete homeostatic chemokines; it is thought that infection‐inflicted destruction of stromal cells results in the downregulation of these chemokines. Whether inflammation per se also regulates these processes remains unclear. We show that TNF accumulated upon non‐infectious immunization of mice similarly downregulates the expressions of these chemokines and consequently diminishes the ingress of naive lymphocytes in inflamed SLOs. Mechanistically, TNF inactivated NIK in LTβR‐stimulated cells and induced the synthesis of Nfkb2 mRNA encoding p100; these together potently accumulated unprocessed p100, which attenuated the RelB activity as inhibitory IκBδ. Finally, a lack of p100 alleviated these TNF‐mediated inhibitions in inflamed SLOs of immunized Nfkb2 −/− mice. In sum, we reveal that an inhibitory TNF‐p100 pathway modulates the adaptive compartment during immune responses.

B-cell memory was long characterized as isotype-switched, somatically mutated and germinal centre (GC)-derived. However, it is now clear that the memory pool is a complex mixture that includes unswitched and unmutated cells. Further, expression of CD73, CD80 and CD273 has allowed the categorization of B-cell memory into multiple subsets, with combinatorial expression of the markers increasing with GC progression, isotype-switching and acquisition of somatic mutations. We have extended these findings to determine whether these markers can be used to identify IgM memory phenotypically as arising from T-dependent versus T-independent responses. We report that CD73 expression identifies a subset of antigen-experienced IgM+ cells that share attributes of functional B-cell memory. This subset is reduced in the spleens of T-cell-deficient and CD40-deficient mice and in mixed marrow chimeras made with mutant and wild-type marrow, the proportion of CD73+ IgM memory is restored in the T-cell-deficient donor compartment but not in the CD40-deficient donor compartment, indicating that CD40 ligation is involved in its generation. We also report that CD40 signalling supports optimal expression of CD73 on splenic T cells and age-associated B cells (ABCs), but not on other immune cells such as neutrophils, marginal zone B cells, peritoneal cavity B-1 B cells and regulatory T and B cells. Our data indicate that in addition to promoting GC-associated memory generation during B-cell differentiation, CD40-signalling can influence the composition of the unswitched memory B-cell pool. They also raise the possibility that a fraction of ABCs may represent T-cell-dependent IgM memory.

The induction of CD80 on podocytes has been shown in animal models of podocyte injury and in certain cases of nephrotic syndrome. In a lipopolysaccharide (LPS)-induced mouse model of albuminuria, we have recently shown a signalling axis of LPS-myeloid cell activation-TNFα production-podocyte CD80 induction-albuminuria. Therefore, in this report, we investigated the cellular and molecular consequences of TNFα addition and CD80 expression on cultured podocytes. A murine podocyte cell line was used for TNFα treatment and for over-expressing CD80. Expression and localization of various podocyte proteins was analysed by reverse transcriptase-polymerase chain reaction, western blotting and immunofluorescence. HEK293 cells were used to biochemically characterize interactions. Podocytes treated with LPS in vitro did not cause CD80 upregulation but TNFα treatment was associated with an increase in CD80 levels, actin derangement and poor wound healing. Podocytes stably expressing CD80 showed actin derangement and co-localization with Neph1. CD80 and Neph1 interaction was confirmed by pull down assays of CD80 and Neph1 transfected in HEK293 cells. Addition of TNFα to podocytes causes CD80 upregulation, actin reorganization and podocyte injury. Overexpressed CD80 and Neph1 interact via their extracellular domain. This interaction implies a mechanism of slit diaphragm disruption and possible use of small molecules that disrupt CD80-Neph1 interaction as a potential for treatment of nephrotic syndrome associated with CD80 upregulation.

Immunological memory comprising of antigen-specific B and T cells contributes to the acquisition of long-term resistance to pathogens. Interactions between CD40 on B cells and CD40L on T cells are responsible for several aspects of acquired immune responses including generation of memory B cells. In order to gain insights into events leading to memory B cell formation, we analyzed the genome-wide expression profile of murine naive B cells stimulated in the presence of anti-CD40. We have identified over 8,000 genes whose expression is altered minimally 1.5-fold at least at one time point over a 3-day time course. The array analysis indicates that changes in expression level of maximum number of these genes occur within 24 h of anti-CD40 treatment. In parallel, we have studied the events following CD40 ligation by examining the expression of known regulators of naive B cell to plasma cell transition, including Pax5 and BLIMP1. The expression profile of these regulatory genes indicates firstly, that CD40 signaling activates naive B cells to a phenotype that is intermediate between the naive and plasma cell stages of the B cell differentiation. Secondly, the major known regulator of plasma cell differentiation, BLIMP1, gets irreversibly downregulated upon anti-CD40 treatment. Additionally, our data reveal that CD40 signaling mediated BLIMP1 downregulation occurs by non-Pax5/non-Bcl6 dependent mechanisms, indicating novel mechanisms at work that add to the complexity of understanding of B cell master regulatory molecules like BLIMP1 and Pax5. J. Cell. Physiol. 229: 1387–1396, 2014. © 2014 Wiley Periodicals, Inc.

While a number of extrinsic factors are known to promote the survival of plasma cells (PCs), the signaling intermediates involved remain poorly characterized. Here we identified inducible nitric oxide synthase (iNOS) as an intermediate that supported the survival of PCs. PCs deficient in iNOS (Nos2(-/-) PCs) showed enhanced death in vitro, after transfer into congenic adoptive hosts, and in chimeras made with wild-type and Nos2(-/-) bone marrow. The iNOS-mediated protection involved activation of protein kinase G and modulation of endoplasmic reticulum stress components. Activation of caspases was also diminished. We found that iNOS was required for PCs to respond to some prosurvival mediators associated with bone marrow stromal cells and that at least one mediator, interleukin 6, fed directly into this pathway by inducing iNOS.

Susceptibility to DSS induced colitis has been shown to depend on various host and microbial factors, the caveat being that most of these studies have used gene-deficient or gnotobiotic mouse models. In order to study specific factors contributing to colitis susceptibility under more physiological conditions we used inbred mouse strains that differ in their IgA responses but are otherwise healthy. We found that the IgA high strain CBA/CaJ (CBA) is resistant to DSS-colitis unlike the IgA low strain C57BL/6 (B6) strain and resistance correlated with extensive IgA-coating of fecal bacteria and lower fecal bacterial loads. Additionally, we found a more robust intestinal barrier in CBA mouse, along with lower intestinal inflammation and bacterial translocation during colitis induction. Through homologous fecal microbial transplants we found that the IgA-uncoated fraction of B6 flora was relatively more colitogenic. Further, experiments with F1 and F1xParent ‘backcrossed’ progeny generated separately with CBA and B6 dams indicated that pups born to IgA high proximal dams were resistant to colitis. These results indicate a role of passive maternal IgA and active IgA in colitis resistance. In order to understand whether early exposure to passive IgA alone can confer resistance to colitis, we foster-nursed B6 pups on CBA dams and vice-versa but it did not have an effect on DSS susceptibility. Interestingly, oral administration of immunoglobulins to B6 adults did decrease DSS associated disease. Collectively, our data indicate that higher gut IgA in adult life plays a more important role as compared to maternal IgA received during early life in conferring resistance to DSS colitis.

CD40 signaling during B cell activation is known to inhibit terminal differentiation and promote memory generation. Blimp-1 is essential for efficient plasma cell (PC) generation, and although CD40 signaling is known to inhibit Blimp-1 induction during B cell activation, the mechanisms involved have been unclear. We report that CD40 signaling induces miR-125b that targets Blimp-1 transcripts, and increases amounts of the ubiquitin ligase Hrd1 that targets BLIMP-1 protein for proteasomal degradation. CD40 signaling also inhibits the early unfolded protein response (UPR) of activated B cells that precedes the induction of terminal differentiation, and Hrd1 feeds into this pathway by targeting the core UPR component IRE-1α. Strikingly, CD40 signaling in the absence of BCR- or TLR-ligation also repressed Blimp-1 transcripts, suggesting that noncognate ligation of CD40 via T–B interactions may repress Blimp-1 in vivo. In support of this, we find that naive B cells purified from CD40–CD154 interaction–deficient mice express higher amounts of Blimp-1 and lower amounts of microRNAs and Hrd1. Higher basal amounts of Blimp-1 in naive CD40−/− B cells correlate with an increased tendency of the cells to undergo terminal differentiation upon LPS stimulation. Conversely, a 24-h exposure to CD40 ligation during LPS stimulation of wild-type B cells is sufficient to inhibit PC generation. The data show that CD40-mediated inhibition of PC generation is via engagement of multiple pathways that involve repression of Blimp-1 and inhibition of the UPR that prepares cells to become professional secretors. They also show that constitutive CD40 signaling in vivo involving bystander T–B interactions can calibrate B cell differentiation outcomes.

Transient albuminuria induced by pathogen-associated molecular patterns (PAMPs) in mice through engagement of Toll-like receptors (TLRs) is widely studied as a partial model for some forms of human nephrotic syndrome (NS). In addition to TLRs, CD80 has been shown to be essential for PAMP-mediated albuminuria. However, the mechanistic relationships between TLRs, CD80 and albuminuria remain unclear. Here, we show that albuminuria and CD80-uria induced in mice by many TLR ligands are dependent on the expression of TLRs and their downstream signalling intermediate MyD88 exclusively in hematopoietic cells and, conversely, on CD80 expression exclusively in non-hematopoietic cells. TNFα is crucial for TLR-mediated albuminuria and CD80-uria, and induces CD80 expression in cultured renal podocytes. IL-10 from hematopoietic cells ameliorates TNFα production, albuminuria and CD80-uria but does not prevent TNFα-mediated induction of podocyte CD80 expression. Chitohexaose, a small molecule originally of parasite origin, mediates TLR4-dependent anti-inflammatory responses, and blocks TLR-mediated albuminuria and CD80-uria through IL-10. Thus, TNFα is a prominent mediator of renal CD80 induction and resultant albuminuria in this model, and small molecules modulating TLR-mediated inflammatory activation might have contributory or adjunct therapeutic potential in some contexts of NS development., Summary: Systemic TNFα mediates myeloid cell and podocyte cross-talk to cause LPS-induced mouse microalbuminuria, a partial model of human nephrotic syndrome, pointing to potential adjunct therapeutic approaches.

The mitochondrial flavoprotein Aif facilitates murine thymocyte development by reducing oxidative stress., Apoptosis-inducing factor (Aif) is a mitochondrial flavoprotein that regulates cell metabolism and survival in many tissues. We report that aif-hypomorphic harlequin (Hq) mice show thymic hypocellularity and a cell-autonomous thymocyte developmental block associated with apoptosis at the β-selection stage, independent of T cell receptor β recombination. No abnormalities are observed in the B cell lineage. Transgenes encoding wild-type or DNA-binding–deficient mutant Aif rectify the thymic defect, but a transgene encoding oxidoreductase activity–deficient mutant Aif does not. The Hq thymic block is reversed in vivo by antioxidant treatment, and Hq T but not B lineage cells show enhanced oxidative stress. Thus, Aif, a ubiquitous protein, serves a lineage-specific nonredundant antiapoptotic role in the T cell lineage by regulating reactive oxygen species during thymic β-selection.

Macrophages and polymorphonuclear cells (PMNs) rapidly respond to microbial and immune inflammatory stimuli and die during these responses. We have shown earlier that many macrophage and PMN functions are compromised in x-linked immunodeficient (Xid) mice with functional deficiency in Bruton's tyrosine kinase (Btk). We now report that Btk-deficient macrophages show enhanced susceptibility to apoptotic death on exposure to the microbial and immune inflammatory signals bacterial lipopolysaccharide (LPS) and interferon-gamma (IFNγ) in vitro. In vivo in mixed bone marrow (BM) chimeras Btk deficiency leads primarily to loss of peripheral macrophage numbers without affecting BM development, suggesting a role of inflammation-induced apoptosis in regulating macrophage life span. Surprisingly, Btk deficiency does not affect macrophage apoptosis induced by DNA damage or CD95 engagement. Reactive nitrogen and oxygen species also do not contribute to inflammation-induced apoptosis, but apoptotic process involves loss of mitochondrial potential, shows increased activation of caspase 9 and enhanced loss of Bcl-xL. The lack of pro-survival signaling through the Btk-phosphotidylinositol 3-kinase-Akt pathway, and persistent MEK signaling, lead to enhanced death in Btk-deficient macrophages only downstream of inflammatory triggers. These data underline the complex role of Btk in the regulation of macrophage survival and function.

B cells responding to cognate Ag in vivo undergo clonal expansion that is followed by differentiation into Ab-secreting plasma cells or into quiescent restimulable memory. Both these events occur in the germinal center and require that cells exit from proliferation, but the signals that lead to one or the other of these mutually exclusive differentiation pathways have not been definitively characterized. Previous experiments have shown that signals transduced through the TNFRs CD27 and CD40 at the time of B cell stimulation in vitro or in vivo can influence this cell fate decision by inhibiting terminal differentiation and promoting memory. In this study, we show that the PIQED domain of the cytoplasmic tail of murine CD27 and the adapter molecule TNFR-associated factor 2 are involved in this effect. Using pharmacological inhibitors of signaling intermediates, we identify JNK as being necessary and sufficient for the observed inhibition of terminal differentiation. While JNK is involved downstream of CD40, inhibition of the MEK pathway can also partially restore plasma cell generation, indicating that both signaling intermediates may be involved. We also show that inhibition of induction of IFN regulatory factor 4 and B lymphocyte induced maturation protein 1 are downstream events common to both receptors.

To compare immune phenotypes across two geographic and ethnic communities, we examined umbilical cord blood by flow cytometry and Luminex in parallel cohorts of 53 newborns from New Delhi, India, and 46 newborns from Stanford, California. We found that frequencies of a B cell subset suggested to be B-1-like, and serum IgM concentration were both significantly higher in the Stanford cohort, independent of differences in maternal age. While serum IgA levels were also significantly higher in the Stanford cohort, IgG1, IgG2, and IgG4 were significantly higher in the New Delhi samples. We found that neutrophils, plasmacytoid dendritic cells, CD8+ T cells, and total T cells were higher in the U.S. cohort, while dendritic cells, patrolling monocytes (CD14dimCD16+), natural killer cells, CD4+ T cells, and naïve B cells were higher in the India cohort. Within the India cohort, we also identified cell types whose frequency was positively or negatively predictive of occurrence of infection(s) in the first six months of life. Monocytes, total T cells, and memory CD4+ T cells were most prominent in having an inverse relationship with infection. We suggest that these data provide impetus for follow-up studies linking phenotypic differences to environmental versus genetic factors, and to infection outcomes.

Germ-line retroviral insertions in vertebrate genomes are implicated in the modulation of host immune responses. We demonstrate that CBA/J mice, which carry the proviral integrants mammary tumor virus locus 6 (Mtv6) and mammary tumor virus locus 7 (Mtv7), are less resistant to infection with the protozoan pathogen Leishmania major compared with closely related but Mtv6-negative and Mtv7-negative CBA/CaJ mice. Although both strains generated comparable L. major-specific CD4 T cell frequencies, T cells from CBA/J mice made much less interferon γ (IFN-γ). L. major-infected CBA/CaJ dendritic cells primed L. major-specific and allospecific IFN-γ-producing CD4 T cells better in vivo and in vitro, respectively, than CBA/J dendritic cells did. L. major susceptibility appeared to be associated with Mtv7, and v-Sag-7 superantigen expression and L. major infection together reduced the ability of an antigen-presenting cell line to prime alloresponder CD4 T cells for IFN-γ commitment. These data show that an endogenous superantigen can interact with L. major infection to alter antigen-presenting cell properties and modulate T cell cytokine commitment, with implications for human susceptibility to infectious diseases.

Poor T cell immunity is one of the many defects seen in elderly humans and aged (Ad) mice. We report that naive CD4 T cells from aged mice (ANCD4 cells) showed greater apoptosis upon primary activation than those from young (Yg) mice, with loss of mitochondrial membrane potential, poor activation of Rel family transcription factors and increased DNA damage. Their ability to enhance glycolysis, produce lactate and induce autophagy following activation was also compromised. ANCD4 cells remained susceptible to death beyond first cell division. Activated ANCD4 cells also showed poor transition to a 'central memory' (CM) CD44(high), CD62L(high) phenotype in vitro. This correlated with low proportions of CM cells in Ad mice in vivo. Functionally, too, IFN-gamma responses recalled from T cells of immunized Ad mice, poor to begin with, worsened with time as compared with Yg mice. Thus, ANCD4 cells handle activation-associated stress very poorly due to multiple defects, possibly contributing to poor formation of long-lasting memory.

HIV-1 Nef has been reported to disrupt MHC class II (MHCII)-mediated Ag presentation by a dual strategy that comprises a reduction in cell surface levels of peptide-loaded mature MHCII molecules and a up-regulation of immature MHCII molecules. We show that Nef achieves relocation of MHCII away from the cell surface in monocytic cells by both delaying its transport to the cell surface and by accelerating endocytic removal of cell surface MHCII to a lysosomal compartment. Nef-induced MHCII endocytosis is cholesterol-sensitive but clathrin- and dynamin-independent. Internalized MHCII molecules traverse the early endosomal system and colocalize with pinocytic cargo before reaching lysosomes. Nef-triggered MHCII endocytosis requires Rab5 activity and lyst function, whereas lysosomal trafficking of internalized MHCII molecules requires Rab7 activity. We further show that a similar pathway can remove peptide-MHCII complexes from the surface of monocytic cells not expressing Nef. Our data suggest that Nef uses mechanisms involved in normal MHCII recycling and turnover to mediate the delivery of cell surface MHCII to a lysosomal destination. Thus, Nef-mediated endocytosis of MHCII provides a novel perspective on the regulation of normal MHCII trafficking.

The heat shock protein (HSP) Hsp90 is known to chaperone cytosolic peptides for MHC class I (MHCI)-restricted antigen presentation to T lymphocytes. We now demonstrate a role for Hsp90 activity in presentation of antigens on MHCII. Treatment of mouse antigen-presenting cells (APC) with the pharmacological Hsp90 inhibitor, geldanamycin, inhibited MHCII-mediated presentation of endocytosed and cytosolic proteins as well as synthetic peptides to specific T cells. Ectopic expression of human Hsp90 in APC enhanced MHCII-mediated antigen presentation. Further, pharmacological Hsp90 inhibition reduced, while retroviral Hsp90 overexpression enhanced, the levels of stable compact MHCII heterodimers correlating with the antigen presentation phenotype. Pharmacological inhibition of Hsp90 activity in IFN-gamma-treated APC resulted in severe abrogation of MHCII-restricted presentation of cytosolic antigen, but only partially inhibited exogenous antigen presentation. Our data suggest a major role for Hsp90 activity in MHCII-mediated antigen presentation pathways, and implicate IFN-gamma-inducible Hsp90-independent mechanisms.

Antigen-presenting cells (APCs) are expected to present peptides from endocytosed proteins via major histocompatibility complex (MHC) class II (MHCII) molecules to T cells. However, a large proportion of peptides purified from MHCII molecules are derived from cytosolic self-proteins making the pathway of cytosolic peptide loading onto MHCII of critical relevance in the regulation of immune self-tolerance. We show that peptides derived from cytoplasmic proteins either introduced or expressed in the cytoplasm are first detectable as MHCII-peptide complexes in LAMP-1+ lysosomes, prior to their delivery to the cell surface. These peptide-MHC complexes are formed in a variety of APCs, including peritoneal macrophages, dendritic cells, and B cells, and are able to activate T cells. This process requires invariant chain (Ii)-dependent sorting of MHCII to the lysosome and the activity of the molecular chaperone H-2M. This pathway is independent of the ER resident peptide transporter complex TAP and does not take place by cross-presentation from neighbouring cells. In conjunction with our earlier results showing that these peptides are derived by cytosolic processing via the proteasome, these observations provide evidence for a ubiquitous route for peptide transport into the lysosome for the efficient presentation of endogenous and cytoplasmic proteins to CD4 T cells.

Clearance of facultative intracellular pathogens such as Salmonella requires IFN-γ from CD4 T cells. Mechanisms linking intracellular pathogen recognition with induction of IFN-γ-producing T cells are still poorly understood. We show in this study that IL-12 is not required for commitment to the IFN-γ-producing T cell response in infection with Salmonella typhimurium, but is needed for its maintenance. The IL-12-independent signals required for commitment depend on events during the first hour of infection and are related to Ag presentation. Even transient attenuation of Ag presentation early during infection specifically abrogates the IFN-γ component of the resulting CD4 T cell response. The IL-12 needed for maintenance is also better induced by live rather than dead bacteria in vivo, and this difference is due to specific suppression of IL-12 induction by dead bacteria. Presence of exogenous IL-4 down-modulates IL-12 production by macrophages activated in vitro. Furthermore, macrophages from IL-4-null mice secrete high levels of both IL-12 and IL-18 in response to stimulation in vivo even with dead bacteria, but this does not lead to induction of IFN-γ-secreting T cells in response to immunization with dead S. typhimurium. Early IL-4 is contributed by triggering of CD4 NK T cells by dead, but not live, bacteria. Thus, Ag presentation-related IL-12-independent events and IL-4-sensitive IL-12-dependent events play crucial complementary roles in the generation of the IFN-γ-committed CD4 T cell component of the immune response in Salmonella infection.

Macrophages from X-linked immunodeficient (xid) mice lacking functional Bruton’s tyrosine kinase (Btk) show poor NO induction and enhanced IL-12 induction, and contribute to delayed clearance of injected microfilaria (mf) in vivo. We now show that Btk is involved in other macrophage effector functions, such as bactericidal activity and secretion of proinflammatory cytokines (TNF-α, IL-1β), but not the T cell-directed cytokine IL-12. Induction of some transcriptional regulators of the NF-κB family, crucial for the expression of proinflammatory cytokines, is also poor in Btk-deficient macrophages. Thus, Btk appears to be involved in signaling for inducible effector functions, but not APC functions, in macrophages. Furthermore, adoptive transfer of T cells from mf-infected xid or wild-type mice did not alter the course of mf clearance in recipients, mf clearance was unaltered in IFN-γ-deficient mice, and improved mf clearance was seen only if greater inducibility of IL-12 was accompanied by greater NO secretion from macrophages, as seen in Ityr C.D2 mice as compared with congenic Itys BALB/c mice. Thus, delayed mf clearance in xid mice was correlated not with the high IL-12/Th1 phenotype but with low NO induction levels. Also, xid macrophages showed poor toxicity to mf in vitro as compared with wild-type macrophages. Inhibition of NO production blocked this mf cytotoxicity, and an NF-κB inhibitor blocked both NO induction and mf cytotoxicity. Thus, Btk is involved in inducing many macrophage effector functions, and delayed mf clearance seen in Btk-deficient xid mice is due to poor NO induction in macrophages, resulting in compromised microfilarial toxicity.

Naïve CD4 T (NCD4T) cells post-activation undergo programming for inducible production of cytokines leading to generation of memory cells with various functions. Based on cytokine based polarization of NCD4T cells in vitro, programming for either 'Th1' (interferon-gamma [IFNg]) or 'Th2' (interleukin [IL]-4/5/13) cytokines is thought to occur via mutually exclusive expression and functioning of T-bet or GATA-3 transcription factors (TFs). However, we show that a high proportion of mouse and human memory-phenotype CD4 T (MCD4T) cells generated in vivo which expressed either Th1 or Th2 cytokines commonly co-expressed T-bet and GATA-3. While T-bet levels did not differ between IFNg-expressing and IL-4/5/13-expressing MCD4T cells, GATA-3 levels were higher in the latter. These observations were also confirmed in MCD4T cells from FVB/NJ or aged C57BL/6 or IFNg-deficient mice. While MCD4T cells from these strains showed greater Th2 commitment than those from young C57BL/6 mice, pattern of co-expression of TF was similar. Effector T cells generated in vivo following immunization also showed TF co-expression in Th1 or Th2 cytokine producing cells. We speculated that the difference in TF expression pattern of MCD4T cells generated in vivo and those generated in cytokine polarized cultures in vitro could be due to relative absence of polarizing conditions during activation in vivo. We tested this by NCD4T cell activation in non-polarizing conditions in vitro. Anti-CD3 and anti-CD28-mediated priming of polyclonal NCD4T cells in vitro without polarizing milieu generated cells that expressed either IFNg or IL-4/5/13 but not both, yet both IFNg- and IL-4/5/13-expressing cells showed upregulation of both TFs. We also tested monoclonal T cell populations activated in non-polarizing conditions. TCR-transgenic NCD4T cells primed in vitro by cognate peptide in non-polarizing conditions which expressed either IFNg or IL-4/5/13 also showed a high proportion of cells co-expressing TFs, and their cytokine commitment varied depending on genetic background or priming conditions, without altering pattern of TF co-expression. Thus, the model of mutually antagonistic differentiation programs driven by mutually exclusively expressed T-bet or GATA-3 does not completely explain natural CD4 T cell priming outcomes.

Reports indicate that, variously, 10–50% of bacteria in the gut are coated with IgA and that bacterial diversity correlates with IgA amounts. In support of these observations, it was recently shown that there is an overall reduction in bacterial diversity in Rag1−/− mice. In extension to these findings, we report that taxa that are severely depleted in Rag1−/− mice include Proteobacteria and SFB, which are IgA-coated in wild-type (WT) mice. We reasoned that IgA might be required for these taxa to take up residence in the gut. Further, since germ-free SCID mice can be stably colonized with SFB, we explored the possibility that retention of SFBs may require continuous exposure to the organisms. We tested this by co-housing Rag1−/− mice with WT mice and found that by day 5, Rag1−/− mice had acquired both Proteobacteria and SFB. In addition, when F2 littermates obtained from WT x Rag1−/− crosses were compared for microbial diversity, both were present in the Rag1−/− pups. These data raise the possibility that mice are being colonized continuously. We also report that members of Proteobacteria separate equally into IgA-bound and IgA-unbound fraction, raising the possibility that the IgA-unbound fraction may represent new colonizers. Finally, in agreement with earlier reports, which showed that transfer of IgA-coated bacteria increases the susceptibility to DSS-colitis in germ-free mice, we find that Rag1−/− mice, which lack coated bacteria, are more resistant to DSS-colitis.

Functional heterogeneity in apparently homogenous T cell populations has been well reported. However, what factors lead to such variations within a T cell population is still unclear. Depending upon the time of egress from the thymus and duration of residence in the periphery, individual cells would be of relatively different ages, however, consequences of such cellular aging have not been addressed. We observed that naive CD8 T cells from aged mice have lower coreceptor levels than naive CD8 T cells of young mice. Further, when naive T cells from young mice are separated into coreceptor-hi and coreceptor-lo subsets, we find that coreceptor-lo cells are smaller, respond poorly and are more susceptible to death. Similar coreceptor-lo associated defects were observed even in TCR-transgenic monoclonal CD8 T cell population. However, this phenotype is an outcome of peripheral residence, as coreceptor-lo and coreceptor-hi subsets of single-positive (SP) thymocytes do not show these differences. We show that coreceptor levels reduce over time, as a result of MHC-I mediated tonic signals and lower CD8 levels can be used as a surrogate marker to identify relatively aged cells in a naive CD8 T cell population. Thus, our data show that apparently unimodally distributed coreceptor heterogeneity among naive T cells, simplistically expected to be due to intrinsic noise, is not only correlated with major functional variation but is modulated by extrinsic microenvironmental cues in vivo. These findings indicate that ensemble averages may not represent individual cell function, even for a unimodal normal distribution of single cell measurements, highlighting the importance of distinguishing between population versus individual T cell responses to antigen encounter. At the population level, CD8 T cell response magnitude increases with ligand dose as well as with ligand affinity. However, whether ligand density and affinity only modulate the frequency of responding T cells and/or alters responses at the single cell level is unclear. We observed that the early activation response of individual naive CD8 T cell is binary such that the frequency of CD69 expressing cells increases with increment in ligand density or affinity, but the levels of CD69 expression in individual responding cells remain constant and variations in baseline cellular metabolic status between individual T cells may contribute to the differences in their activation thresholds. We show that ligand density and affinity determine the extent of TCR internalization in responding CD8 T cells, which in turn regulates the time to cell cycle entry. TCR internalization, which is commonly thought of as a mechanism to attenuate TCR-mediated signaling, was found to be associated with sustained TCR signaling via PI3K, leading to rapid metabolic reprogramming and when the degradation of internalized TCR complex is prevented, the duration of signaling is extended possibly from endosomal compartments, leading to better T cell response. Collectively, our findings reveal that heterogeneity among T cells for signal receptivity as well as microenvironmental influences experienced during their life history shape individual T cell responses and understanding the basis of heterogeneity within putatively homogenous T cell populations is significant for understanding of immune T cell responses. Citation Format: Renu Balyan, Rupali Gund, Chitra Ebenezer, Thyagarajan Krishnamurthy, Amanpreet Singh Chawla, Jeannine Durdik, Anna George, Vineeta Bal, Satyajit Rath. Functionally significant heterogeneity in apparently homogenous naive CD8 T cell populations. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A80.

Following Japanese encephalitis virus (JEV) infection neutralizing antibodies are shown to provide protection in a significant proportion of cases, but not all, suggesting additional components of immune system might also contribute to elicit protective immune response. Here we have characterized the role of T cells in offering protection in adult mice infected with JEV. Mice lacking α/β–T cells (TCRβ–null) are highly susceptible and die over 10–18 day period as compared to the wild-type (WT) mice which are resistant. This is associated with high viral load, higher mRNA levels of proinflammatory cytokines and breach in the blood-brain-barrier (BBB). Infected WT mice do not show a breach in BBB; however, in contrast to TCRβ-null, they show the presence of T cells in the brain. Using adoptive transfer of cells with specific genetic deficiencies we see that neither the presence of CD4 T cells nor cytokines such as IL-4, IL-10 or interferon-gamma have any significant role in offering protection from primary infection. In contrast, we show that CD8 T cell deficiency is more critical as absence of CD8 T cells alone increases mortality in mice infected with JEV. Further, transfer of T cells from beige mice with defects in granular lytic function into TCRβ-null mice shows poor protection implicating granule-mediated target cell lysis as an essential component for survival. In addition, for the first time we report that γ/δ-T cells also make significant contribution to confer protection from JEV infection. Our data show that effector CD8 T cells play a protective role during primary infection possibly by preventing the breach in BBB and neuronal damage., Author summary Japanese encephalitis virus (JEV) commonly infects human beings in developing countries including those in Southeast Asia. While the majority of the infected people suffer from mild illness, a minority suffers from encephalitis which may lead to death. The virus is transmitted by mosquito bites and elimination of mosquitoes is not a practical answer to prevent the disease, therefore, prevention by vaccination is a desired goal. While various vaccines are clinically tried and some are marketed further improvement in vaccines is still possible. In a complex disease like JE many components of the immune system contribute to variable extent in protection. We show here that one subset of T cells called CD8 cells which are capable of killing infected cells are very critical for providing protection against JEV infection in mice. In the absence of T cells we also observed that virus reaches the brain early, unlike in the presence of T cells, and this possibly results in high virus load in the brain leading to worsening of the condition and death. Thus, our data help in identifying the role of CD8 T cells in protection from lethal JEV infection and the information may be useful for modifying and/or developing vaccine for prevention of JEV-mediated disease.