bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA breaks in immunoglobulin Class Switch Recombination that depend on UNG but not AID
Authors: Max Edward E. , Arudchandran Arulvathani , Bernstein Ralph M.
Published: June 2021

B lymphocytes switch from secreting IgM to secreting IgG, IgA or IgE through a DNA recombination, Class Switch Recombination. CSR is thought to be triggered by Activation Induced Deaminase (AID), which is believed to deaminate cytosines to uracil in Switch region DNA. Excision of uracils by uracil DNA glycosylase (product of the UNG gene) generates abasic sites, which are targeted for the DNA cleavage required for CSR. Consistent with this model, CSR‐related double‐strand breaks (dsbs) – detected by Ligation Mediated PCR (LMPCR) ‐‐ have been reported to be dramatically reduced in either AID−/− or UNG−/− B‐cells. Here we examine single‐strand breaks (ssbs) using LMPCR and report, surprisingly, that CSR‐related antisense‐strand breaks in Sμ and Sγ regions are dependent on UNG but not AID, suggesting participation of a cytosine deaminase other than AID. This conclusion is supported by the sequences at these DNA breaks, which show a bias for a consensus sequence different from that reported for AID. The ssbs appear to be part of the normal CSR pathway, since in B cells in which CSR is blocked by deletion of Sμ, the content of Sγ ssbs is elevated, as though ssbs persist in the absence of a recombination partner for completing μ‐to‐γ CSR. These results suggest a narrower role for AID in CSR than previously recognized, and prompt a search for a putative alternative cytosine deaminase participating in CSR.

Supported by FDA funds.
Differential Histone H3 Profile at the Promoter of Vasoactive Intestinal Peptide Receptor‐1 in B and T cells
Authors: Benton Keith David , Hermann Rebecca , Vomhof‐Dekrey Emilie , Dorsam Glenn
Published: June 2021

Post‐translational modifications of histones can regulate transcription. Histone 3 (H3) modifications, such as the addition of an acetyl group, contributes to an accessible chromatin state. Acetylation at H3K9 and H3K14 are correlated with a transcriptionaly permissive environment. The anti‐proliferative G Protein coupled receptor (GPCR), vasoactive intestinal peptide receptor‐1 (VPACR‐1), is highly expressed in naïve CD4 T‐cells, downregulated 80% in activated CD4 T cells, and undetected in B cells. We hypothesize that high VPACR‐1 expression in naïve T cells versus activated T cells and B cells is directly related to an elevated promoter H3 acetylation pattern. To test this hypothesis, splenic CD4 T and B cells were analyzed by Chromatin Immunoprecipitation (ChIP) with antibodies against mono‐acetylated (H3K9) or diacetylated (H3K9/K14) residues, and analyzed by qPCR. In support of our hypothesis, there was a significant enrichment for H3K9 acetylation in T versus B cells at the VPACR‐1 promoter. Surprisingly, in activated CD4 T cells, the H3K9 acetylation was maintained along with a concomitant enrichment in diacetylation (H3K9/K14). In summary, there appears to be differential H3 acetylation at the VPACR‐1 promoter in T and B lymphocytes, but its expression profile may not directly correlate with its epigenetic landscape. Funding: 1KO1 DK064828; COBRE 2P20RR015566.
Aberrant T cell KIR demethylation in the pathogenesis of lupus
Authors: Liu Ying , Basu Dhiman , Strickland Faith M , Richardson Bruce
Published: June 2021

Human lupus is an epigenetic disease. Some lupus‐inducing drugs are DNA methylation inhibitors, and T cell DNA demethylation causes lupus in mice through aberrant expression of genes causing macrophage (Mø) apoptosis and B cell overstimulation. Identical T cell DNA demethylation contributes to human lupus by overexpression of the same molecules. Molecules expressed on demethylated but not normal T cells, and which inactivate the pathologic T cells, would be ideal targets for treating lupus. We treated human T cells with a DNA methylation inhibitor, identified overexpressed genes with microarrays, and selected the KIR gene family from the >300 genes induced. KIR genes are normally expressed on NK but not T cells, and deliver inhibitory or stimulatory signals depending on the gene. KIR promoter demethylation was confirmed by bisulfite sequencing and function by cassette methylation. Crosslinking the inhibitory KIR prevented Mø killing by demethylated cells. T cells from lupus patients lupus aberrantly expressed the KIR family, and crosslinking inhibitory KIR blocked autologous Mø killing. Anti‐KIR may be an effective treatment for human lupus. Supported by PHS grants AR42525, AG25877 and ES015214.
Combination of KIR‐HLA genotypes implicated in weak inhibition and autoimmunity augments risk for Birdshot Chorioretinopathy in HLA‐A29 positive individuals
Authors: Rajalingam Raja , Levinson Ralph D
Published: June 2021

HLA‐A29 is strongly associated with birdshot chorioretinopathy (BCR), a chronic ocular inflammatory disease with characteristic choroidal lymphocytic infiltrates. Although HLA‐A29 occurs frequently in all populations, BCR affects only a small percentage of HLA‐A29 positive Caucasians, indicating additional susceptibility factors for BCR. Discovery of HLA class I‐specific killer cell immunoglobulin‐like receptors (KIR) led to a series of epidemiological studies implicating KIR‐HLA gene combinations in disease. Here, we characterized KIR‐HLA pairs in BCR patients and controls carrying HLA‐A29 as well as controls lacking HLA‐A29. We found that combination of KIR‐HLA genotypes implicated for weak inhibition (KIR2DL2/3+HLA‐C1 and KIR3DL1+HLA‐Bw4T80) and autoimmunity (KIR2DS2/S3/S4) augments the risk of developing BCR in HLA‐A29 positive individuals. The reciprocal association of strong inhibitory pairs (KIR3DL1+HLA‐Bw4I80 and KIR2DL1+HLA‐C2) in combination with those implicated in protection from infection (KIR3DS1+HLA‐Bw4I80 and KIR2DS1+HLA‐C2) was observed in HLA‐A29‐negative controls.
Generation of T‐ and IgA‐secreting B cells in the murine large intestine is independent of retinoic acid
Authors: Cha Hyeran , Chang Sun‐Young , Kweon Mi‐Na
Published: June 2021

Retinoic acid (RA), the vitamin A (retinol) metabolite, generates gut‐homing specific T‐ and IgA‐secreting B cells in the small intestine (SI). We found that generation of T‐ and polyclonal IgA‐secreting B cells in the mouse large intestine (LI) are independent of RA. In contrast to the SI, the numbers of IgA+B cells and CD4+ or CD8+ T cells in the lamina propria of LI were not changed in mice fed a vitamin A‐deficient (−) diet. Further, the numbers of Ag‐specific IgA Ab‐secreting cells (ASC) in the LI induced by intrarectal vaccination with tetanus toxoid and cholera toxin were identical in mice fed vitamin A (+) and vitamin A (−) diets, whereas significant reduction of Ag‐specific IgA ASC was detected in the SI of mice fed vitamin A (−) diets after oral vaccination. Interestingly, in mice treated orally with 50 μg of RA from 1 week before oral immunization, the numbers of Ag‐specific IgA ASC were dramatically decreased in the lamina propria of the SI. However, the numbers of Ag‐specific IgA ASC were normally induced in the lamina propria of the LI with or without RA treatment before intrarectal vaccination. These data demonstrate that generation and/or homing of T‐ and IgA‐secreting B cells in the LI are independently regulated by RA.
TSLP‐TSLPR interactions license dendritic cell responses and CD4+ T cell dependent immunity and inflammation in the intestine
Authors: Taylor Betsy C , Zaph Colby , Troy Amy E , Du Yurong , Guild Katherine , Artis David
Published: June 2021

Thymic stromal lymphopoeitin (TSLP) is an epithelial cell derived cytokine that has been shown to promote Th2 cytokine‐dependent inflammation in the skin and lung. However, the role of TSLP in regulating Th2 cytokine‐dependent immunity and inflammation in the intestine has not been tested. Utilizing TSLPR−/− mice we showed that TSLP‐TSLPR interactions are critical for immunity to the intestinal pathogen Trichuris. While littermate controls exhibited expression of Th2 cytokines and cleared infection, TSLPR−/− mice produced increased levels of Trichuris‐specific IFNγ and remained persistently infected. In vitro, pre‐exposure of DCs to rTSLP selectively inhibited LPS‐induced IL‐12/23p40 expression suggesting that TSLP functions to inhibit proinflammatory cytokines. To address whether TSLP‐TSLPR interactions were necessary for the induction of the protective Th2 cytokine response following Trichuris infection, TSLPR−/− mice were infected and treated with neutralizing anti‐IFNγ antibodies. Following anti‐IFNγ treatment, TSLPR−/− mice exhibited a protective Th2 immune response and cleared infection. Taken together, these data suggest that within the intestine TSLP‐TSLPR interactions are not required to directly promote pathogen‐specific Th2 cytokine responses, but instead create a Th2 permissive environment through inhibition of DC‐derived proinflammatory cytokines.
Preferential Loss of Th17 T‐cells at Mucosal Sites Predicts AIDS Progression in Simian Immunodeficiency Virus‐Infected Macaques
Authors: Trindade Christopher Julius , Heraud Jean‐Michel , Cecchinato Valentina , Laurence Arian , Brenchley Jason , Tryniszewska Elzbieta , Ferrari Maria Grazia , Tsai Wen‐Po , Vaccari Monica , Washington‐Parks Robyn , Douek Daniel C. , O'shea John , Franchini Genoveffa
Published: June 2021

Depletion of CD4+ T‐cells in the gut is necessary but not sufficient to cause AIDS in animal models, raising the possibility that differential depletion of CD4+ T‐cell subtypes may be important. We found that Th17 cells, a recently identified lineage of effector CD4+ T‐helper interleukin‐17‐secreting cells, express CCR5, are infected in vitro and in vivo, and are preferentially depleted within a few weeks in the pathogenic model of simian immunodeficiency virus (SIVmac251) infection of macaques. Importantly, their frequency is not restored to normal levels over time. We also found that SIVmac251‐infected “elite controllers” maintain normal levels of Th17 T‐cells in all tissues, and that there is a negative correlation between Th17 cell frequency at mucosal sites and plasma virus level, suggesting their importance in HIV/SIV pathogenesis. Because Th17 cells play a central role in innate and adaptive immune response to extracellular bacteria, these data provide a rational explanation for the chronic enteropathy in HIV infection. Thus, therapeutic approaches to incease Th17 numbers and/or function may be of benefit in HIV infection.
Influence of IgA expression on T‐Dependent and T‐Independent antibody responses
Authors: Kirimanjeswara Girish Soorappa , Salmon Sharon , Metzger Dennis W
Published: June 2021

Individuals with selective IgA deficiency are competent in producing IgG antibodies but often suffer from recurrent respiratory infections with encapsulated bacteria. We examined the immunological basis for this hyper‐susceptibility using an IgA deficient (IgA KO) mouse model. Following i.m. vaccination, WT and IgA KO mice produced similar levels of all antibody isotypes (other than IgA) against T‐dependent antigens, including CRM197 and Influenza HA and NA. However, IgA KO mice were severely defective in producing IgG antibody against T‐independent (TI) antigens such as polysaccharides, polysaccharides conjugated to carrier proteins, although they did produce normal levels of IgM. Inoculation of IL‐12 as an adjuvant during vaccination increased IgG antibody levels in both wild‐type and IgA KO mice to the same relative degree, suggesting a quantitative rather than qualitative defect in TI antigen‐responsive cells in IgA KO mice. Indeed, flow cytometric analyses revealed that IgA KO mice contained significantly fewer peritoneal B‐1a B cells, the cell type thought to be primarily responsible for TI antibody responses. These results could explain the frequent failure of conjugate vaccines to induce protective immunity in IgA‐deficient patients. Further investigations are underway to elucidate the molecular basis for the defective class switching by IgA KO mice in response to TI antigens.

RO1 AI41715
Memory CD4+ T cells are refractory to TGF‐beta‐mediated, retinoic acid‐enhanced conversion into Foxp3+ regulatory T cells by oral antigen
Authors: Kim Byung‐Seok , Park Young‐Jun , Kim Yun‐Sun , Kim Yeon‐Jeong , Shim Doo‐Hee , Kweon Mi‐Na , Kang Chang‐Yuil
Published: June 2021

Although it is well known that orally administered antigen can efficiently induce peripheral tolerance to prevent otherwise serious autoimmune diseases, most studies have failed to ameliorate the ongoing diseases by oral antigen. To clarify the cause of resistance to oral tolerance in subjects with an active autoimmune state, we focused on oral antigen‐induced generation of CD4+Foxp3+ regulatory T cells (Tregs) which play a crucial role in oral tolerance. Here, we show that Ag‐specific memory CD4+ T cells are refractory to oral antigen‐induced conversion into Tregs, whereas Tregs can easily be generated from naïve CD4+ T cells by oral antigen. We also show that Ag‐specific memory CD4+ T cells are inferior to naïve CD4+ T cells in TGF‐β‐mediated conversion into Tregs in vitro, although all‐trans retinoic acids (ATRA) can enhance the conversion of them. In addition, when we compared CD45RBlow or CD62Llowmemory phenotype(MP) CD4+T cells with CD45RBhigh or CD62Lhigh CD4+ naïve cells in TGF‐β‐mediated, ATRA‐enhanced conversion in vitro, MP CD4+ T cells were less succeptible to TGF‐β‐mediated Treg induction and were totally refractory to ATRA‐mediated enhancement of conversion. Altogether, these findings suggest that the refractoriness of memory CD4+ T cells to oral antigen‐induced conversion into Tregs can be the cause of unresponsiveness to oral tolerance in patients with active autoimmune diseases.
Identification of vaccine candidate peptides and harvesting epitopes of Brucella melitensis
Authors: Durward Marina , Harms Jerome , Peltz Carrie , Sidney John , Sette Alessandro , Splitter Gary
Published: June 2021

Brucella spp. is a facultative intracellular bacteria that is considered a Class B Select Agent and the available human vaccines are ineffective in producing immunological memory. Adaptive immune responses are crucial to the control of Brucella, as previous studies have shown that the acute response is CD8+ T cell mediated. Also, there are no defined immunogenic epitopes for any Brucella, and this is a critical hole in our understanding of how to control this bacterial infection. BALB/c mouse Class I alleles, H2Kd and H2Dd, were used in RankPep, a peptide prediction algorithm, to predict Class I Brucella peptides. After analysis of the data generated from each individual Brucella ORF, a group of peptides were chosen as probable high affinity binders. These peptides were synthesized and then tested in a competitive MHC binding assay. Pooled peptide vaccinations and subsequent quantification of immune activation by intracellular cytokine staining is a high‐throughput method for investigating the roles of large numbers of predicted Class I peptides. Preliminary results demonstrate that 22.5% of the predicted peptides are naturally presented during Brucella melitensis infection of BALB/c mice. These data present an important first step in the development of a multivalent peptide vaccine that will protect against brucellosis. Supported by NIH 1R01AI073558‐01A1 and GLRCE 1U54AI057153.
Early CD8 T cell proliferative heterogeneity entails diverse memory differentiation programs
Authors: Kalia Vandana , Sarkar Surojit , Kaech Susan , Ahmed Rafi
Published: June 2021

The lineage of memory cells is a key issue that is much debated. While several lines of evidence support the notion that memory cells are direct descendants of effector cells, data suggesting that memory cells do not pass through a fully differentiated effector phase also exist. Recently a tantalizing notion that divergent memory and effector cell fates are decided after the first cell division has also been presented. Considering the fact that initial antigenic encounter programs CD8 T cells to enter a mode of extensive proliferation, we hypothesized that in the setting of an ongoing acute infection events transpiring beyond the first round of cell division are likely to impact cell fate decisions. In this study we performed a detailed analysis of CD8 T cell differentiation with respect to cell division following activation and also evaluated the memory generation potential of CD8 T cells in early or late rounds of cell division. Our studies demonstrate that as cells continue to proliferate beyond the first round of division, they acquire potent effector functions in a largely homogenous manner. While fraction of effector CD8 T cells in both early late rounds of cell division persisted into memory phase, memory cells derived from effector cells in early rounds of cell division preferentially upregulated L‐selectin and localized in the lymph nodes compared to memory cells derived from effector cells in later rounds of cell division. This also correlated with more robust recall proliferation associated with memory cells derived from effector cells in early rounds of cell division. Collectively, these studies present a model that heterogeneity in initial proliferation, putatively from differential priming events, regulates differentiation of memory cells.
The interplay between dendritic cell subsets and T cells during peripheral virus infection
Authors: Carbone Francis R. , Wakim Linda , Gebhardt Thomas , Heath William R.
Published: June 2021

Dendritic cells are a heterogenous population consisting of a number of distinct subsets. It is likely that this heterogeneity reflects functional differences between the populations. We have been exploring the role of dendritic cells during infection with herpes simplex virus (HSV) ‐ starting with the initial virus infection in the skin through to the reactivation of HSV within the nerves. I will present results of experiments examining how different dendritic cell subsets are called into play during the separate phases of infection with this virus.
Viral meningitis in real time
Authors: McGavern Dorian B. , Kang Silvia S. , Kim Jiyun , Dustin Michael L.
Published: June 2021

Meningitis is a potentially fatal disorder induced by a long list of human pathogens. We therefore propose that a detailed understanding of this pathogenic process in real time may foster the development of novel interventions to prevent permanent neurological dysfunction / fatalities. Intracerebral inoculation of mice with LCMV Armstrong (Arm) induces fatal meningitis within 6 days that is mediated by cytotoxic lymphocytes (CTL). The precise mechanisms that mediate seizures and fatal injury during virus‐induced meningitis are not known. To gain advanced mechanistic insights into immune cell dynamics and function in the virally infected central nervous system (CNS), we examined disease pathogenesis in real time using intravital two photon microscopy. Our real time studies revealed highly dynamic immune cell activity in the meningeal space of LCMV‐infected mice, which resulted in profound blood barrier breakdown. Our studies also provide direct evidence that CTL divide within the CNS and that innate immune cells (e.g., microglia) respond rapidly to point sources of CNS damage. Finally, direct injection of anti‐MHC I antibody into the subarachnoid space interfered with the activities of virus‐specific CTL. We therefore propose that examination and antagonism of immune cells through a “window on the brain” should rapidly advance our understanding of CNS viral pathogenesis.
An anti‐viral immune response clears virally infected neurons from the mouse brain through cytotoxic mechanisms involving IFN‐γ, perforin, and TNFα.
Authors: Lowenstein Pedro R , Bergeron Josee , Zirger Jeffery , Barrett Robert , Liu Chunyan , Castro Maria G
Published: June 2021

The mechanisms by which the immune system eliminates virally infected cells from the brain remain unknown. We utilized a replication‐defective adenovirus to avoid direct viral‐induced cytotoxicty. A marker gene (HSV1‐TK) expressed from the adenoviral vector was used to label, characterize and quantitate infected mouse brain cells. Upon systemic immunization against adenovirus, a CD4+ and CD8+ T cell‐dependent immune response reduced the number of infected cells by >50%. Using specific knockout mice we determined that IFNy, perforin and TNFa, were necessary for eliminating virally infected cells. To demonstrate whether the immune system eliminates transgene expression through killing of transduced cells, or functional inhibition of expression from the viral genome, we developed a method that simultaneously measures gene expression from infected cells, and the number of infected cells' genomes. Using ROSA26 mice and adenoviral vectors expressing Cre, we demonstrated that the immune system abolished expression from both the viral genome and the cellular genome. Our results suggest that CD4+ and CD8+ T cells eliminate viral gene expression from the brain through cytotoxic mechanisms involving IFN‐γ, perforin, and TNFα.

This work was funded by grants from NINDS, NIH.
Role of type I interferon in the Theiler's virus‐induced encephalitis, cellular infiltration to the CNS and function of immune cells
Authors: JIN YOUNG‐HEE , Hou Wanqiu , Fuller Alyson C. , Kang Bongsu , Kim Byung S.
Published: June 2021

Theiler's murine encephalomyelitis virus (TMEV), a single stranded RNA virus, causes a persistent infection in the central nervous system (CNS) and an immune‐mediated demyelination similar to human multiple sclerosis. To examine the role of type I interferon (IFN‐I)‐mediated signals in TMEV infection, mice lacking a subunit of the type I IFN receptor (IFN‐IR KO mice) were utilized. In contrast to the wildtype 129S2/SP mice unaffected after intracerebral infection with TMEV, IFN‐IR KO mice developed rapid fetal encephalitis accompanied with high viral load. IFN‐IR KO mice showed greater infiltration of immune cells to the CNS. Because of the higher levels of infiltration, the overall numbers of virus‐specific, IFN‐γ‐producing T cells were higher, despite the lower proportions of the virus‐specific infiltrating cells. Interestingly, altered expression patterns of MHC and co‐stimulatory molecules were observed in antigen presenting cells of IFN‐IR KO mice, leading to the cytokine production pattern of T cells differing from that of the wildtype control mice. These results suggest that IFN‐I‐mediated signals play important roles in cellular infiltration to the CNS and shaping protective immune responses, in addition to modulating viral replication. (Supported by RO1 NS28752, RO1 NS33008, RG 3392‐A5)
CD8 collapse results in CD4 deficits and Disease Course Acceleration in SIV‐infected Rhesus Macaques
Authors: Marcondes Maria Cecilia G , Sopper Sieghart , Sauermann Ulrike , Burdo Tricia H , Watry Debbie , Zandonatti Michelle , Fox Howard
Published: June 2021

In SIV‐infected monkeys, defects in memory CD4+ T cells correlate with disease development. However, the events leading to these deficits are unknown. Examining animals with a rapid disease course, we find an initial development, followed by collapse, of SIV‐specific CTL. This decrease is accompanied by global changes in CD8 cells, and occurs in synchrony with the characteristic CD4 deficiencies. Antibody‐mediated depletion of CD8 cells early after SIV infection induces similar changes in the CD4 cells and rapid development of AIDS. Thus CD8 collapse results in the development of a defective and insufficient CD4 population. Our results indicate that a breakdown in CD8 cells results in the CD4 deficits leading to disease.
Herpes Simplex Virus 2 infection up‐regulates TLRs expression and activates NF‐kB in human cervical epithelial cells
Authors: Li Hui , Tan Yun , Wei Yun , Zuo Zehua , Wu Xinxing
Published: June 2021

Toll‐like receptors (TLRs) play a crucial role in the innate immune response against microbial infections, engaging differential signaling pathways, leading to activation of T cell and therefore triggering acquired immunity. Herpes Simplex Virus type 2 (HSV‐2) is one of the most common sexually transmitted pathogen and can establish lifelong latency infection. HSV‐2 is a significant co‐factor in the transmission and acquisition of HIV‐1. To determine the effect of HSV‐2 infection on TLRs expression pattern and to investigate the TLR‐mediated innate immune reponse to HSV‐2 in human cervical cells, we infected immortalized human cervical epithelial cells (HECs) with HSV‐2 (G strain) as a model in this study. The results showed that normal HECs expressed TLR1, 2, 3, 4,5, 6,9 and HSV‐2 infection up‐regulated TLRs expression at mRNA and protein level. TLR9 was dramatically elevated at late phage 36hr post‐infection by Real‐time RT‐PCR analysis. The NF‐kB and MAP Kinase p38 were rapidly activated as early as 1hr post‐infection. Different proinflammatory cytokines and interferons were also determined during the course of HSV‐2 infection. Thus, HECs possess the ability to sensor HSV‐2 infection and activate the host innate immune response through TLRs.

This work was supported by grants from the National Natural Science Foundation of China (No. 30500465) and China Wuhan Chenguang Science Foundation.
mPDCA‐1 Ag reveals a novel antigen presenting cell modulating Th2 type immune responses against influenza virus infection
Authors: Yoo Jae‐Kwang , Fish Eleanor
Published: June 2021

Objectives:

Here, we have identified a novel immune cell population expressing mPDCA‐1 Ag in lymphoid tissues, which are mPDCA1+CD11c−TcRß−B220−. Studies were conducted to investigate the in vivo function of this cell population in the context of influenza virus infection.

Methods:

B6 mice were infected with A/WSN/33 influenza virus by intranasal inoculation. The subsequent cellular responses of the mPDCA1+CD11c−TcRß−B220− cells were monitored by FACS‐analysis.

Results:

Study revealed that these cells are APCs responding to viral infection. Pulmonary infection with A/WSN/33 virus stimulated these cells to up‐regulate the surface expression of MHC‐II and co‐stimulatory molecules. Influenza virus infection induced the migration of this cell into infected lungs, spleen and DLN. In the DLN, mPDCA1+CD11c−TcRß−B220− cells presented viral antigens to CD4+ T cells. Influenza virus activated mPDCA1+CD11c−TcRß−B220− cells also produced cytokines including IL‐25, IL‐2 and IL‐4 in lungs and DLN. Notably, ex vivo studies revealed that influenza virus activated mPDCA1+CD11c−TcRß−B220− cells induced the generation of Th2 effector T cells from antigen primed CD4+ T cells in the DLN.

Conclusion:

These results suggest that a newly described mPDCA1+CD11c−TcRß−B220− cell population may regulate a Th2 type immune response against pulmonary influenza virus infection.
Examining age‐related function and phenotype of influenza‐specific CD8+T cells in humans
Authors: Dolfi Douglas V , Polley Antonio M , Wherry E John
Published: June 2021

Development of severe infection and secondary complications, despite previous exposure to or vaccination against influenza virus, leads to increased mortality in the elderly. Improved vaccines, designed based on understanding of the differences in the immune responses of elderly people as compared to young people, inducing universal protection to all influenza virus strains are needed. We have used several approaches to examine phenotype and function of influenza‐specific CD4+ and CD8+ T cells as well as the breadth of the T cell response to influenza virus between young and aged individuals. Using MHC class I tetramers we determined relationships between the total number of influenza‐specific CD8+ T cells and their ability to produce effector cytokines, or proliferate, upon in vitro restimulation to examine whether there are differences in CD8+ T cells from aged and young individuals. We are examining how functional aspects of CD4+ and CD8+ T cells from young and aged individual correlate with inhibitory receptor and cell senescence marker expression. We will also evaluate the ability of inhibitory and costimulatory pathways to modify those functions. Other differences in phenotype and function of antiviral T cells between young and elderly individuals must be examined to determine how these differences may affect the quality of virus‐specific immunity in aged individuals.
Defining the phenotype, function and differentiation state of CD4 T cells during chronic infection
Authors: Crawford Alison , Wherry E. John
Published: June 2021

The immune system evolved to provide protection against a wide range of pathogens. Under some circumstances, however, the immune response is ineffective. During many chronic viral infections the antigen‐specific T cells become ‘exhausted’, allowing viral persistence. Exhaustion has been described for CD8 T cells during a number of viral infections including the human infections HIV and HCV, and the chronic LCMV infection in mice. However, despite the central role CD4 T cells play in directing immune responses their function and differentiation during chronic infections is not well understood. We have defined the exhaustion of CD4 T cells during chronic LCMV, both in terms of phenotype and function. As with CD8 T cells, CD4 T cells upregulated several inhibitory receptors, though some are distinct to the different T cell subsets. Moreover, CD4 T cells show a hierarchical loss of function, though the loss of cytokine production may be more severe than for CD8 T cells. In addition, we examined other CD4 T cell functions such as provision of help. Thus, these studies help in unraveling the complexity of CD4 T cell function during chronic infection.
A distinct role for T‐bet in the maintenance of antigen‐specific CD8 T‐cells during chronic viral infection
Authors: Kao Charlly Chuan , Intlekofer Andrew M. , Reiner Steven L. , Wherry E. John
Published: June 2021

The T‐box transcription factor, T‐bet, has a well‐established role as a master regulator of CD4 T cell commitment to the TH1 lineage; however, its importance in the regulation of CD8 differentiation has only recently begun to be elucidated. T‐bet, in cooperation with another highly homologous T‐box family member, eomesodermin, is necessary for induction of the cytokine, interferon‐γ, and the expression of genes integral to the cytolytic machinery, such as granzyme and perforin. Inflammatory stimuli, including IL‐12, can potentiate its expression in cytotoxic T cells after antigenic activation. While T‐bet contributes to the induction of CD8 effector function during acute viral infections, excessive T‐bet expression may impede the ability to differentiate into long‐lived memory cells, instead favoring the formation of short‐lived fully differentiated effector T cells. We studied the expression and function of T‐bet in mice infected with the chronic clone‐13 strain of the lymphocytic choriomeningitis virus (LCMV) and compared the response to that observed with the acute Armstrong strain. Surprisingly, we find that CD8 T cells express lower levels of T‐bet during clone‐13 compared to Armstrong infection even though they are exposed to an environment with persisting antigen and heightened inflammation. This may contribute to the dysfuction (i.e. “exhaustion”) of CD8 T cells observed in chronically infected mice, as T‐bet knockout mice have higher viral burden and lose their antigen‐specific cells within weeks post‐infection. Thus, T‐bet plays a complex role during CD8 differentiation and can have vastly different effects depending on the context of the infection.
The role of CD4 T cell help in restoring function of exhausted CD8 T cells during chronic infection
Authors: Aubert Rachael D , Sarkar Surojit , Ahmed Rafi
Published: June 2021

Exhaustion of CD8 T cell function is a hallmark of chronic viral infections. Although much advance has been made in understanding the mechanisms mediating this dysfunction, little is known about the functional role of CD4 T cell help in this process. In the current study we investigated the effect newly primed antigen specific CD4 T cells may have on CD8 T cell exhaustion and disease outcome in the murine model of chronic viral infection LCMV Clone‐13. We show that adoptive transfer of monoclonal CD4 T cells during the late stages of an ongoing chronic infection can potentially restore function of virus specific CD8 T cells and lead to reduced viral levels. Furthermore, we demonstrate that blocking the PD‐1 (Programmed Death Recptor‐1) inhibitory pathway following the CD4 T cell transfer results in a synergistic effect in terms of both functional rescue of CD8 T cells and viral control. Our data provides new evidence that delivery of CD4 T cell help during ongoing chronic infection may lead to restoration of CD8 T cell function and allow favorable disease outcome.

Funding for this work is through NIH grants to R. Ahmed.
Correlates of protection efficacy induced by VACV‐specific CD8+ T cell epitopes in the murine intranasal challenge model
Authors: Moutaftsi Magdalini , Salek‐Ardakani Shahram , Croft Michael , Grey Howard , Sette Alessandro
Published: June 2021

Vaccinia virus (VACV) was used to eradicate smallpox and is considered the gold standard of vaccines. Elucidating the immunobiology of protection will reveal vaccinology principles that can be applied to future vaccine development. The identification of 49 VACV‐specific CD8+ T cell epitopes offers a unique opportunity to systematically analyze the correlation between protective efficacy and the characteristics of T cell responses.

All epitopes were tested for their ability to protect peptide‐immunized C57Bl/6 mice from lethal intranasal challenge with VACV‐WR. The epitopes varied significantly in their capacity to confer protection; 15 were 75–00% protective, 22 were 25–5% protective and 12 were less than 25% protective. Measurement of IFN‐ γ following peptide immunization demonstrated that induction of high IFN‐γ correlated with high protection efficacy, whereas low IFN‐γt confer any protection. However, for some epitopes the IFN‐γtion did not correlate with protective efficacy. Ongoing studies aim to identify the underlying mechanisms that induce protective immunity by characterizing the T cell responses elicited by epitope immunization and infection.

These issues are relevant for the development of new vaccine constructs, in which inclusion of isolated antigens associated with detrimental responses could jeopardize vaccine efficacy.

Grant Support: NO1‐AI40024
Rapid Induction of Autophagy by Infection of Human Monocytes with Yersinia pestis
Authors: Tournier Jean‐Nicolas , Adrian Scott , Hakami Ramin , Ruthel Gordon , Ulrich Robert G
Published: June 2021

Autophagy is a regulated cellular process for recycling the cell‘s own cytoplasm under extreme conditions of starvation and infection. Activation of autophagy triggers a double membrane to wrap around pathogens during cellular infection, forming an autophagosome organelle that sequentially fuses with late endosomes and lysosomes. The light chain (LC)3 (or Atg‐8) that drives the formation of the vacuole is one of several autophagy‐related proteins (Atg proteins) that regulate the process of autophagy. LC3 is found in two forms: LC3‐I is cytosolic while LC3‐II is conjugated to ethanolamine and is bound to autophagosomes. We infected human monocytes with Yersinia pestis strain CO92 (Pgm–, Pla–) and then analyzed cells by confocal microscopy and Western blot for the expression of LC3 protein. Y. pestis infection rapidly induced LC3‐I expression within 15 min. The expression of LC3‐II, the form found in the inner part of autophagosome‐forming vacuoles, peaked at 2 hours. The peak expression of LC3‐II coincided with the first appearance of bacteria within lysosomes. Moreover, the expression of LC3 was dependent on the temperature of Y. pestis pre‐culture, suggesting that temperature‐modulated Y. pestis pathogenicity may interfere with autophagy cell defense processes.
Direct upregulation of human CD4+ T cell proliferation and cytokine secretion by Mycobacterium tuberculosis (MTB)
Authors: Lancioni Christina Louise , Thomas Jeremy , Ding Xuedong , Pecora Nicole D. , Drage Michael , Harding Clifford , Boom W. Henry , Rojas Roxana E.
Published: June 2021

Apart from modulating antigen presenting cell functions, recent studies suggest that MTB can directly regulate CD4+ T cell function. The current study investigates the regulatory role of MTB molecule(s) on CD4+ T cell activation in the absence of antigen presenting cells. Highly pure (99.8%) CD4+ T cells were obtained from peripheral blood of healthy PPD‐ negative donors by negative selection with mAb coated‐ magnetic beads followed by flow sorting. MTB (H37Ra) lysate was extracted with Triton X‐114; hydrophobic material was separated by SDS‐PAGE and electroelution (MTB fractions). CD4+ T cells were stimulated with suboptimal concentrations of plate‐bound anti‐CD3 plus soluble anti‐CD28 mAbs or fibronectin +/− different MTB fractions. Significant increases in CD4+ T‐cell proliferation, IL‐2‐ and IFN‐gamma secretion in response to anti‐CD3/anti‐CD28 were observed in presence of MTB fractions containing low‐molecular weight bands (<20 kDa). Up‐regulation of proliferation and IFN‐gamma was also observed in presence of MTB fractions upon stimulation with anti‐CD3 plus fibronectin. MTB fractions did not induce T cell activation when incubated in absence of TCR stimulation and a second co‐stimulatory signal (anti‐CD28 or fibronectin). Thus hydrophobic MTB fractions have a direct co‐stimulatory effect on CD4+ T cells suggesting a role of MTB in direct regulation of T cell activation.
Persistent pulmonary inflammation and cyclooxygenase ‐1 and ‐2 modifications in alveolar macrophages following in vivo phagocytosis of mycobacteria
Authors: Shibata Yoshimi , Henriksen Ruth Ann , Myrvik Quentin N , Patel Harni , Pavlovic Mirjana D. , Shinohara Shizuka , Pantuso Traci , Shinohara Tsutomu
Published: June 2021

Granuloma formation is a hallmark of mycobacterial infections and represents the histological correlate of both protective immunity and inflammatory tissue destruction. PGE2 is known to regulate infections, inflammation and tissue repair, although exact mechanisms regulating COX‐2 activity, a rate‐limiting enzyme for PGE2 biosynthesis, are unclear. In this study, alveolar macrophages (MØ) were isolated from C57Bl/6 mice following intranasal administration of 0.5 mg killed Mycobacteirum bovis BCG. We found that normal alveolar MØ showed minimal (or undetectable) levels of COX‐1 and COX‐2 by western blot. However, one day after administration, almost all MØ had phagocytosed BCG and expressed significant levels of COX‐1 and COX‐2. Analysis by confocal microscopy indicated that for MØ phagocytosing BCG, COX‐1 and COX‐2 were preferentially dissociated from the nuclear envelop (NE), indicating forms catalytically inactive for PGE2 synthesis. In contrast to mice given ip BCG where peritoneal MØ containing BCG disappeared within 7 days, 28 days after intranasal administration, 68% of alveolar MØ in lung lavage fluid still contained BCG together with NE‐dissociated COX‐1 and COX‐2. Our results indicate that the lack of PGE2 production may enhance PGE2‐sensitive, MØ c‐mediated immune responses against pulmonary infections and inflammation caused by mycobacteria. [NIH HL71711 and DOD DAMD17‐03‐1‐0004]
Elevated cyclic AMP Acts through PKA type I to Inhibit Mycobacterium tuberculosis‐induced IFN‐γ Secretion by T cells
Authors: Chung Yoontae , Barnes Peter F. , Wang Xisheng , Townsend James C. , Pasquinelli Virginia , Jurado Javier O. , Garcia Veronica E. , Weis Stephen , Samten Buka
Published: June 2021

IFN‐γ secretion is reduced in human tuberculosis (TB), but the underlying mechanisms are unclear. Intracellular cAMP in PBMC from TB patients was higher (1182±93 fmol, n=12) than that in healthy tuberculin reactors (499±91 fmol, n=7, p<0.001), as measured by ELISA, and cAMP levels correlated inversely with M. tb‐induced IFN‐γ production (r=−0.535, p< 0.05). Using selective cAMP agonists, cAMP‐mediated inhibition of M. tb‐induced IFN‐γ secretion by PBMC from healthy tuberculin reactors was found to depend on PKA type I but not PKA type I II or EPAC (early exchange protein directly activated by cAMP). When electrophoretic mobility shift assays were performed to evaluate binding of nuclear extracts of M. tb‐stimulated PBMC to the IFN‐γ proximal promoter, only the PKA type cAMP analogue markedly inhibited M. tb‐induced DNA‐binding activity of the transcription factors, cAMP response element binding protein (CREB), activating transcription factor (ATF)‐2 and c‐Jun, which positively regulate IFN‐γ transcription. Western blotting confirmed reduced expression of CREB, ATF‐2 and c‐Jun in cells treated with M. tb and the PKA type I agonist cAMP analogue. We conclude that increased cAMP utilizes the PKA type I pathway to reduce M. tb‐induced IFN‐γ secretion by inhibiting binding of transcription factors to the IFN‐γ proximal promoter. (NIH AI063514)
Delivery of rapamycin to dendritic cells using degradable microparticles
Authors: Jhunjhunwala Siddharth , Raimondi Giorgio , Thomson Angus W. , Little Steven R.
Published: June 2021

Degradable microparticles have the potential to protect and release drugs over extended periods and, if sized appropriately, can be targeted passively to phagocytic cells in vivo. Dendritic cells (DCs) are a class of phagocytic cells that are known to play important roles in transplant rejection. Previously, we have demonstrated that DCs treated with an immunosuppressive drug, rapamycin, have the ability to suppress transplant rejection. We have devised a strategy to deliver an intracellular depot of rapamycin to DCs. To achieve this, rapamycin was encapsulated into ∼3.4 μm sized Poly‐lactic‐co‐glycolic acid (PLGA) microparticles (rapaMPs), and release behavior examined under intra‐phagosomal (pH = 5) and extracellular (pH = 7.4) conditions. We observe that rapaMPs are internalized by DCs in large numbers, and that they do not induce DC death. Further, we demonstrate that the DCs that take up rapaMPs have significantly reduced ability to activate T cells, in comparison to DCs treated with soluble rapamycin. Hence, we conclude that DC specific intracellular delivery of rapamycin results in better efficacy of the drug (with respect to the ability to modulate DC function) when compared to treating DCs with extracellular rapamycin. We are currently in the process of investigating the hypothesis that rapaMP‐loaded DCs can be used to suppress transplant rejection.
Reduction of murine Syngeneic graft‐versus‐host disease (SGVHD) following treatment with the superoxide dismutase mimetic MnTBAP
Authors: Perez Jacqueline , Brandon J. Anthony , Jennings Chester D. , Caywood Betty , Kaplan Alan M , Bryson J. Scott
Published: June 2021

Syngeneic graft‐vs‐host disease (SGVHD) is induced by reconstituting lethally irradiated mice with syngeneic bone marrow followed by a 21 day treatment with the immunosuppressive agent cyclosporine A (CsA). Symptoms of the disease appear 2–3 weeks following cessation of CsA therapy and disease‐associated inflammation occurs primarily in the colon and liver. Irradiation and CsA are both required for the development of SGVHD and both contribute to the production of oxidative stress. Time course studies revealed increased reactive oxygen and nitrogen species in CsA treated animals compared to BMT controls. Studies also demonstrated increased migration of CD4+T cells into the colon during CsA therapy. In addition, increased colon mRNA levels of proinflammatory cytokines (TNF‐α, IL‐12) and cell adhesion molecules (CAMs) were observed as early as day 14‐post BMT in colon of CsA treated animals. Since reactive oxygen species (ROS) are known to mediate CsA toxicity, studies were undertaken to determine the effect of CsA‐induced oxidative stress on the induction of SGVHD. Treatment with the antioxidant MnTBAP, during CsA therapy, caused a reduction in SGVHD development as well as decreased colon mRNA levels for iNOS, CAMs and proinflammatory cytokines. These studies suggest a role for oxidative stress in the development of intestinal pathology associated with murine SGVHD. Research support: NIH Grant P01‐CA092372.
Anti‐CD40LAb combined with CTLA4Ig prolonged porcine islet xenograft survival indefinitely in GalT(+/+) C57BL/6 mice but not indefinitely in syngeneic GalT(−/−) mice
Authors: Cho Hyoung‐Soo , Yoon Il‐Hee , Lim Jong‐Hyung , Kim Yong‐Hee , Kim Jung‐Sik , Kim Sang‐Joon , Park Chung‐Gyu
Published: June 2021

Defining the immune response to xenogeneic pancreatic islet graft and induction of immune tolerance are crucial for the successful islet xenotransplantation. Galactosyltransferase‐deficient(GalT‐KO) mice provide a unique opportunity to study xenograft rejection in the context of anti‐α1,3‐Gal antibody(α‐Gal Ab) response. We investigated the rejection mechanism of porcine islet xenograft in C57BL/6 GalT‐KO mice. 5000 islet equivalent of pig islets isolated from CMS miniature pig were transplanted under the kidney capsule of diabetic GalT‐KO or normal B6 mice. Graft rejection occurred earlier in GalT‐KO(mean survival day(MSD)=7) than in normal mice(MSD=15). Heavy infiltration of CD4+ and CD8+T cells in rejected graft implicated T cells as major player in graft rejection. Notably, intra‐graft infiltration of polymorphonuclear leukocytes with CD4+ T cells in peri‐islet on DPT 3 was observed only in GalT‐KO recipients, which was abolished by CD4+T cell depletion. α‐CD40LmAb+CTLA4Ig prolonged graft survival indefinitely in wild type(>200 days), but not indefinitely in GalT‐KO mice(MSD=60) where the rising titer of α‐Gal Ab was observed. Conclusively, the control of α‐Gal Ab and its subsequent immune response in addition to T cell would be required for immunological tolerance in discordant islet xenotransplantation such as pig to GalT‐KO mice or pig to human islet xenotransplantation.
Human B cells producing allograft‐specific HLA antibodies
Authors: Han Mei , Rogers James , Comeaux Ashley , Stastny Peter
Published: June 2021

Development of donor‐specific HLA antibodies following organ transplantation is associated with acute and chronic rejection. This experiment was designed to investigate recipient B lymphocytes to determine whether HLA antibodies can be produced in vitro. The method is based on culturing peripheral blood B cells stimulated by a cell line expressing CD40 ligand. Purified B cells were obtained from 26 subjects: 13 allograft recipients, 9 sensitized patients on the transplant waiting list, 2 multiparous women with serum HLA antibodies, and 2 healthy non‐transfused males. The culture supernatants were screened for antibodies with Luminex flow cytometry beads coupled with multiple HLA class I or class II antigens. Supernatants with antibodies were further analyzed using single antigen‐coupled beads to determine antibody specificity. Results showed that HLA antibody‐producing B cells were detected in persons known to be sensitized. In 10 of 13 allograft recipients, antibodies against mismatched donor HLA antigens were observed. In two patients we found donor‐specific antibodies produced in B cell cultures when serum reactions were negative. Our results suggest that analysis of supernatants of CD40 ligand‐stimulated B cells using Luminex single antigen HLA beads may be a useful tool for identifying and monitoring the donor‐specific humoral immune response in organ transplant recipients.