Publications by authors named "Jennifer M Dan"

30 Publications

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AI-guided discovery of the invariant host response to viral pandemics.

EBioMedicine 2021 Jun 11:103390. Epub 2021 Jun 11.

Moores Cancer Center, University of California San Diego, USA; Department of Cellular and Molecular Medicine, University of California San Diego, USA; Medicine, University of California San Diego, USA. Electronic address:

Background: Coronavirus Disease 2019 (Covid-19) continues to challenge the limits of our knowledge and our healthcare system. Here we sought to define the host immune response, a.k.a, the "cytokine storm" that has been implicated in fatal COVID-19 using an AI-based approach.

Method: Over 45,000 transcriptomic datasets of viral pandemics were analyzed to extract a 166-gene signature using ACE2 as a 'seed' gene; ACE2 was rationalized because it encodes the receptor that facilitates the entry of SARS-CoV-2 (the virus that causes COVID-19) into host cells. An AI-based approach was used to explore the utility of the signature in navigating the uncharted territory of Covid-19, setting therapeutic goals, and finding therapeutic solutions.

Findings: The 166-gene signature was surprisingly conserved across all viral pandemics, including COVID-19, and a subset of 20-genes classified disease severity, inspiring the nomenclatures ViP and severe-ViP signatures, respectively. The ViP signatures pinpointed a paradoxical phenomenon wherein lung epithelial and myeloid cells mount an IL15 cytokine storm, and epithelial and NK cell senescence and apoptosis determine severity/fatality. Precise therapeutic goals could be formulated; these goals were met in high-dose SARS-CoV-2-challenged hamsters using either neutralizing antibodies that abrogate SARS-CoV-2•ACE2 engagement or a directly acting antiviral agent, EIDD-2801. IL15/IL15RA were elevated in the lungs of patients with fatal disease, and plasma levels of the cytokine prognosticated disease severity.

Interpretation: The ViP signatures provide a quantitative and qualitative framework for titrating the immune response in viral pandemics and may serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs.

Funding: This work was supported by the National Institutes for Health (NIH) [grants CA151673 and GM138385 (to DS) and AI141630 (to P.G), DK107585-05S1 (SD) and AI155696 (to P.G, D.S and S.D), U19-AI142742 (to S.

C, Cchi: Cooperative Centers for Human Immunology)]; Research Grants Program Office (RGPO) from the University of California Office of the President (UCOP) (R00RG2628 & R00RG2642 to P.G, D.S and S.D); the UC San Diego Sanford Stem Cell Clinical Center (to P.G, D.S and S.D); LJI Institutional Funds (to S.C); the VA San Diego Healthcare System Institutional funds (to L.C.A). GDK was supported through The American Association of Immunologists Intersect Fellowship Program for Computational Scientists and Immunologists.

One Sentence Summary: The host immune response in COVID-19.
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http://dx.doi.org/10.1016/j.ebiom.2021.103390DOI Listing
June 2021

Increased peripheral blood neutrophil activation phenotypes and NETosis in critically ill COVID-19 patients: a case series and review of the literature.

Clin Infect Dis 2021 May 14. Epub 2021 May 14.

Division of Pulmonary, Critical Care and Sleep Medicine, Department of Medicine, University of California San Diego (UCSD), La Jolla, CA 92093, USA.

Background: Increased inflammation has been well defined in COVID-19, while definitive pathways driving severe forms of this disease remain uncertain. Neutrophils are known to contribute to immunopathology in infections, inflammatory diseases and acute respiratory distress syndrome (ARDS), a primary cause of morbidity and mortality in COVID-19. Changes in neutrophil function in COVID-19 may give insight into disease pathogenesis and identify therapeutic targets.

Methods: Blood was obtained serially from critically ill COVID-19 patients for eleven days. Neutrophil extracellular trap formation (NETosis), oxidative burst, phagocytosis and cytokine levels were assessed. Lung tissue was obtained immediately post-mortem for immunostaining. Pubmed searches for neutrophils, lung and COVID-19 yielded ten peer-reviewed research articles in English.

Results: Elevations in neutrophil-associated cytokines IL-8 and IL-6, and general inflammatory cytokines IP-10, GM-CSF, IL-1b, IL-10 and TNF, were identified both at first measurement and across hospitalization (p<0.0001). COVID neutrophils had exaggerated oxidative burst (p<0.0001), NETosis (p<0.0001) and phagocytosis (p<0.0001) relative to controls. Increased NETosis correlated with leukocytosis and neutrophilia, and neutrophils and NETs were identified within airways and alveoli in lung parenchyma of 40% of SARS-CoV-2 infected lungs available for examination (2 out of 5). While elevations in IL-8 and ANC correlated with disease severity, plasma IL-8 levels alone correlated with death.

Conclusions: Literature to date demonstrates compelling evidence of increased neutrophils in the circulation and lungs of COVID-19 patients. importantly, neutrophil quantity and activation correlates with severity of disease. Similarly, our data shows that circulating neutrophils in COVID-19 exhibit an activated phenotype with enhanced NETosis and oxidative burst.
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http://dx.doi.org/10.1093/cid/ciab437DOI Listing
May 2021

Differential T cell reactivity to endemic coronaviruses and SARS-CoV-2 in community and health care workers.

J Infect Dis 2021 Apr 2. Epub 2021 Apr 2.

Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla, CA, USA.

Herein we measured CD4 + T cell responses against common cold corona (CCC) viruses and SARS-CoV-2 in high-risk health care workers (HCW) and community controls. We observed higher levels of CCC reactive T cells in SARS-CoV-2 seronegative HCW compared to community donors, consistent with potential higher occupational exposure of HCW to CCC. We further show that SARS-CoV-2 T cell reactivity of seronegative HCW was higher than community controls and correlation between CCC and SARS-CoV-2 responses is consistent with cross-reactivity and not associated with recent in vivo activation. Surprisingly, CCC T cell reactivity was decreased in SARS-CoV-2 infected HCW, suggesting that exposure to SARS-CoV-2 might interfere with CCC responses, either directly or indirectly. This result was unexpected, but consistently detected in independent cohorts derived from Miami and San Diego.
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http://dx.doi.org/10.1093/infdis/jiab176DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8083569PMC
April 2021

Negligible impact of SARS-CoV-2 variants on CD4 and CD8 T cell reactivity in COVID-19 exposed donors and vaccinees.

bioRxiv 2021 Mar 1. Epub 2021 Mar 1.

The emergence of SARS-CoV-2 variants highlighted the need to better understand adaptive immune responses to this virus. It is important to address whether also CD4+ and CD8+ T cell responses are affected, because of the role they play in disease resolution and modulation of COVID-19 disease severity. Here we performed a comprehensive analysis of SARS-CoV-2-specific CD4+ and CD8+ T cell responses from COVID-19 convalescent subjects recognizing the ancestral strain, compared to variant lineages B.1.1.7, B.1.351, P.1, and CAL.20C as well as recipients of the Moderna (mRNA-1273) or Pfizer/BioNTech (BNT162b2) COVID-19 vaccines. Similarly, we demonstrate that the sequences of the vast majority of SARS-CoV-2 T cell epitopes are not affected by the mutations found in the variants analyzed. Overall, the results demonstrate that CD4+ and CD8+ T cell responses in convalescent COVID-19 subjects or COVID-19 mRNA vaccinees are not substantially affected by mutations found in the SARS-CoV-2 variants.
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http://dx.doi.org/10.1101/2021.02.27.433180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7941626PMC
March 2021

Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases.

Cell Rep Med 2021 Feb 26;2(2):100204. Epub 2021 Jan 26.

Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla, CA 92037, USA.

T cells are involved in control of SARS-CoV-2 infection. To establish the patterns of immunodominance of different SARS-CoV-2 antigens and precisely measure virus-specific CD4 and CD8 T cells, we study epitope-specific T cell responses of 99 convalescent coronavirus disease 2019 (COVID-19) cases. The SARS-CoV-2 proteome is probed using 1,925 peptides spanning the entire genome, ensuring an unbiased coverage of human leukocyte antigen (HLA) alleles for class II responses. For HLA class I, we study an additional 5,600 predicted binding epitopes for 28 prominent HLA class I alleles, accounting for wide global coverage. We identify several hundred HLA-restricted SARS-CoV-2-derived epitopes. Distinct patterns of immunodominance are observed, which differ for CD4 T cells, CD8 T cells, and antibodies. The class I and class II epitopes are combined into epitope megapools to facilitate identification and quantification of SARS-CoV-2-specific CD4 and CD8 T cells.
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http://dx.doi.org/10.1016/j.xcrm.2021.100204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837622PMC
February 2021

Differential T cell reactivity to seasonal coronaviruses and SARS-CoV-2 in community and health care workers.

medRxiv 2021 Jan 15. Epub 2021 Jan 15.

Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla, CA 92037, USA.

Herein we measured CD4 T cell responses against common cold corona (CCC) viruses and SARS-CoV-2 in high-risk health care workers (HCW) and community controls. We observed higher levels of CCC reactive T cells in SARS-CoV-2 seronegative HCW compared to community donors, consistent with potential higher occupational exposure of HCW to CCC. We further show that SARS-CoV-2 reactivity of seronegative HCW was higher than community controls and correlation between CCC and SARS-CoV-2 responses is consistent with cross-reactivity and not associated with recent in vivo activation. Surprisingly, CCC reactivity was decreased in SARS-CoV-2 infected HCW, suggesting that exposure to SARS-CoV-2 might interfere with CCC responses, either directly or indirectly. This result was unexpected, but consistently detected in independent cohorts derived from Miami and San Diego.
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http://dx.doi.org/10.1101/2021.01.12.21249683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7814840PMC
January 2021

Immunological memory to SARS-CoV-2 assessed for up to eight months after infection.

bioRxiv 2020 Dec 18. Epub 2020 Dec 18.

Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla, CA 92037, USA.

Understanding immune memory to SARS-CoV-2 is critical for improving diagnostics and vaccines, and for assessing the likely future course of the COVID-19 pandemic. We analyzed multiple compartments of circulating immune memory to SARS-CoV-2 in 254 samples from 188 COVID-19 cases, including 43 samples at ≥ 6 months post-infection. IgG to the Spike protein was relatively stable over 6+ months. Spike-specific memory B cells were more abundant at 6 months than at 1 month post symptom onset. SARS-CoV-2-specific CD4 T cells and CD8 T cells declined with a half-life of 3-5 months. By studying antibody, memory B cell, CD4 T cell, and CD8 T cell memory to SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited distinct kinetics.
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http://dx.doi.org/10.1101/2020.11.15.383323DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7805444PMC
December 2020

Immunological memory to SARS-CoV-2 assessed for up to 8 months after infection.

Science 2021 02 6;371(6529). Epub 2021 Jan 6.

Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla, CA 92037, USA.

Understanding immune memory to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for improving diagnostics and vaccines and for assessing the likely future course of the COVID-19 pandemic. We analyzed multiple compartments of circulating immune memory to SARS-CoV-2 in 254 samples from 188 COVID-19 cases, including 43 samples at ≥6 months after infection. Immunoglobulin G (IgG) to the spike protein was relatively stable over 6+ months. Spike-specific memory B cells were more abundant at 6 months than at 1 month after symptom onset. SARS-CoV-2-specific CD4 T cells and CD8 T cells declined with a half-life of 3 to 5 months. By studying antibody, memory B cell, CD4 T cell, and CD8 T cell memory to SARS-CoV-2 in an integrated manner, we observed that each component of SARS-CoV-2 immune memory exhibited distinct kinetics.
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http://dx.doi.org/10.1126/science.abf4063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919858PMC
February 2021

Comprehensive analysis of T cell immunodominance and immunoprevalence of SARS-CoV-2 epitopes in COVID-19 cases.

bioRxiv 2020 Dec 9. Epub 2020 Dec 9.

T cells are involved in control of SARS-CoV-2 infection. To establish the patterns of immunodominance of different SARS-CoV-2 antigens, and precisely measure virus-specific CD4 and CD8 T cells, we studied epitope-specific T cell responses of approximately 100 convalescent COVID-19 cases. The SARS-CoV-2 proteome was probed using 1,925 peptides spanning the entire genome, ensuring an unbiased coverage of HLA alleles for class II responses. For HLA class I, we studied an additional 5,600 predicted binding epitopes for 28 prominent HLA class I alleles, accounting for wide global coverage. We identified several hundred HLA-restricted SARS-CoV-2-derived epitopes. Distinct patterns of immunodominance were observed, which differed for CD4 T cells, CD8 T cells, and antibodies. The class I and class II epitopes were combined into new epitope megapools to facilitate identification and quantification of SARS-CoV-2-specific CD4 and CD8 T cells.
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http://dx.doi.org/10.1101/2020.12.08.416750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7743077PMC
December 2020

Elongated neutrophil-derived structures are blood-borne microparticles formed by rolling neutrophils during sepsis.

J Exp Med 2021 Mar;218(3)

La Jolla Institute for Immunology, La Jolla, CA.

Rolling neutrophils form tethers with submicron diameters. Here, we report that these tethers detach, forming elongated neutrophil-derived structures (ENDS) in the vessel lumen. We studied ENDS formation in mice and humans in vitro and in vivo. ENDS do not contain mitochondria, endoplasmic reticulum, or DNA, but are enriched for S100A8, S100A9, and 57 other proteins. Within hours of formation, ENDS round up, and some of them begin to present phosphatidylserine on their surface (detected by annexin-5 binding) and release S100A8-S100A9 complex, a damage-associated molecular pattern protein that is a known biomarker of neutrophilic inflammation. ENDS appear in blood plasma of mice upon induction of septic shock. Compared with healthy donors, ENDS are 10-100-fold elevated in blood plasma of septic patients. Unlike neutrophil-derived extracellular vesicles, most ENDS are negative for the tetraspanins CD9, CD63, and CD81. We conclude that ENDS are a new class of bloodborne submicron particles with a formation mechanism linked to neutrophil rolling on the vessel wall.
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http://dx.doi.org/10.1084/jem.20200551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721910PMC
March 2021

Antigen-Specific Adaptive Immunity to SARS-CoV-2 in Acute COVID-19 and Associations with Age and Disease Severity.

Cell 2020 11 16;183(4):996-1012.e19. Epub 2020 Sep 16.

Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology (LJI), La Jolla, CA 92037, USA; Department of Medicine, Division of Infectious Diseases and Global Public Health, University of California, San Diego (UCSD), La Jolla, CA 92037, USA. Electronic address:

Limited knowledge is available on the relationship between antigen-specific immune responses and COVID-19 disease severity. We completed a combined examination of all three branches of adaptive immunity at the level of SARS-CoV-2-specific CD4 and CD8 T cell and neutralizing antibody responses in acute and convalescent subjects. SARS-CoV-2-specific CD4 and CD8 T cells were each associated with milder disease. Coordinated SARS-CoV-2-specific adaptive immune responses were associated with milder disease, suggesting roles for both CD4 and CD8 T cells in protective immunity in COVID-19. Notably, coordination of SARS-CoV-2 antigen-specific responses was disrupted in individuals ≥ 65 years old. Scarcity of naive T cells was also associated with aging and poor disease outcomes. A parsimonious explanation is that coordinated CD4 T cell, CD8 T cell, and antibody responses are protective, but uncoordinated responses frequently fail to control disease, with a connection between aging and impaired adaptive immune responses to SARS-CoV-2.
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http://dx.doi.org/10.1016/j.cell.2020.09.038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7494270PMC
November 2020

AI-guided discovery of the invariant host response to viral pandemics.

bioRxiv 2020 Sep 22. Epub 2020 Sep 22.

We sought to define the host immune response, a.k.a, the "cytokine storm" that has been implicated in fatal COVID-19 using an AI-based approach. Over 45,000 transcriptomic datasets of viral pandemics were analyzed to extract a 166-gene signature using ACE2 as a 'seed' gene; ACE2 was rationalized because it encodes the receptor that facilitates the entry of SARS-CoV-2 (the virus that causes COVID-19) into host cells. Surprisingly, this 166-gene signature was conserved in all ral andemics, including COVID-19, and a subset of 20-genes classified disease severity, inspiring the nomenclatures and signatures, respectively. The signatures pinpointed a paradoxical phenomenon wherein lung epithelial and myeloid cells mount an IL15 cytokine storm, and epithelial and NK cell senescence and apoptosis determines severity/fatality. Precise therapeutic goals were formulated and subsequently validated in high-dose SARS-CoV-2-challenged hamsters using neutralizing antibodies that abrogate SARS-CoV-2•ACE2 engagement. IL15/IL15RA were elevated in the lungs of patients with fatal disease, and plasma levels of the cytokine tracked with disease severity. Thus, the signatures provide a quantitative and qualitative framework for titrating the immune response in viral pandemics and may serve as a powerful unbiased tool to rapidly assess disease severity and vet candidate drugs.

One Sentence Summary: The host immune response in COVID-19.
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http://dx.doi.org/10.1101/2020.09.21.305698DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523116PMC
September 2020

Selective and cross-reactive SARS-CoV-2 T cell epitopes in unexposed humans.

Science 2020 10 4;370(6512):89-94. Epub 2020 Aug 4.

Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology, La Jolla, CA 92037, USA.

Many unknowns exist about human immune responses to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus. SARS-CoV-2-reactive CD4 T cells have been reported in unexposed individuals, suggesting preexisting cross-reactive T cell memory in 20 to 50% of people. However, the source of those T cells has been speculative. Using human blood samples derived before the SARS-CoV-2 virus was discovered in 2019, we mapped 142 T cell epitopes across the SARS-CoV-2 genome to facilitate precise interrogation of the SARS-CoV-2-specific CD4 T cell repertoire. We demonstrate a range of preexisting memory CD4 T cells that are cross-reactive with comparable affinity to SARS-CoV-2 and the common cold coronaviruses human coronavirus (HCoV)-OC43, HCoV-229E, HCoV-NL63, and HCoV-HKU1. Thus, variegated T cell memory to coronaviruses that cause the common cold may underlie at least some of the extensive heterogeneity observed in coronavirus disease 2019 (COVID-19) disease.
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http://dx.doi.org/10.1126/science.abd3871DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574914PMC
October 2020

Targets of T Cell Responses to SARS-CoV-2 Coronavirus in Humans with COVID-19 Disease and Unexposed Individuals.

Cell 2020 06 20;181(7):1489-1501.e15. Epub 2020 May 20.

Center for Infectious Disease and Vaccine Research, La Jolla Institute for Immunology, La Jolla, CA 92037, USA; Department of Medicine, Division of Infectious Diseases and Global Public Health, University of California, San Diego, La Jolla, CA 92037, USA. Electronic address:

Understanding adaptive immunity to SARS-CoV-2 is important for vaccine development, interpreting coronavirus disease 2019 (COVID-19) pathogenesis, and calibration of pandemic control measures. Using HLA class I and II predicted peptide "megapools," circulating SARS-CoV-2-specific CD8 and CD4 T cells were identified in ∼70% and 100% of COVID-19 convalescent patients, respectively. CD4 T cell responses to spike, the main target of most vaccine efforts, were robust and correlated with the magnitude of the anti-SARS-CoV-2 IgG and IgA titers. The M, spike, and N proteins each accounted for 11%-27% of the total CD4 response, with additional responses commonly targeting nsp3, nsp4, ORF3a, and ORF8, among others. For CD8 T cells, spike and M were recognized, with at least eight SARS-CoV-2 ORFs targeted. Importantly, we detected SARS-CoV-2-reactive CD4 T cells in ∼40%-60% of unexposed individuals, suggesting cross-reactive T cell recognition between circulating "common cold" coronaviruses and SARS-CoV-2.
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http://dx.doi.org/10.1016/j.cell.2020.05.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7237901PMC
June 2020

Recurrent group A tonsillitis is an immunosusceptibility disease involving antibody deficiency and aberrant T cells.

Sci Transl Med 2019 02;11(478)

Division of Vaccine Discovery, La Jolla Institute for Immunology (LJI), La Jolla, CA 92037, USA.

"Strep throat" is highly prevalent among children, yet it is unknown why only some children develop recurrent tonsillitis (RT), a common indication for tonsillectomy. To gain insights into this classic childhood disease, we performed phenotypic, genotypic, and functional studies on pediatric group A (GAS) RT and non-RT tonsils from two independent cohorts. GAS RT tonsils had smaller germinal centers, with an underrepresentation of GAS-specific CD4 germinal center T follicular helper (GC-T) cells. RT children exhibited reduced antibody responses to an important GAS virulence factor, streptococcal pyrogenic exotoxin A (SpeA). Risk and protective human leukocyte antigen (HLA) class II alleles for RT were identified. Lastly, SpeA induced granzyme B production in GC-T cells from RT tonsils with the capacity to kill B cells and the potential to hobble the germinal center response. These observations suggest that RT is a multifactorial disease and that contributors to RT susceptibility include HLA class II differences, aberrant SpeA-activated GC-T cells, and lower SpeA antibody titers.
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http://dx.doi.org/10.1126/scitranslmed.aau3776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561727PMC
February 2019

Heart transplantation outcomes for rheumatic heart disease: Analysis of international registry data.

Clin Transplant 2018 12 22;32(12):e13439. Epub 2018 Nov 22.

Division of Infectious Diseases and Global Public Health, University of California, San Diego, California.

Background: Rheumatic heart disease (RHD), an autoimmune sequela of Group A streptococcal infection, is a chronic valvular disease affecting 32 million people worldwide, predominantly in developing nations. As the predisposition to autoimmune sequela still remains post transplantation, our primary objective was to assess if there were differences in mortality and rejection rates.

Methods And Results: Using the International Society for Heart and Lung Transplantation (ISHLT) adult heart transplant registry, we identified 42 RHD patients who had undergone heart transplantation between 1988 and 2014. We matched the 42 RHD recipients by transplant year, age, and gender to 420 dilated cardiomyopathy (DCM) recipients. One-year mortality in the RHD group was 17.95% vs. 7.92% in the DCM group (P = 0.07). Survival was significantly reduced in the RHD group vs. the DCM group via Kaplan Meier curves (P = 0.04). In a multivariate model, RHD status (OR 3.19, 95% CI 1.15-8.83, P = 0.025) and serum creatinine (OR 1.41, 95% CI 1.09-1.82, P = 0.009) were associated with an increased odds of one-year mortality (P = 0.0013).

Conclusions: At one year post transplantation, RHD recipients had a significantly lower survival than DCM recipients. RHD status was also an independent predictor of mortality at 1 year post transplantation.
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http://dx.doi.org/10.1111/ctr.13439DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6384093PMC
December 2018

Comparative analysis of activation induced marker (AIM) assays for sensitive identification of antigen-specific CD4 T cells.

PLoS One 2017 24;12(10):e0186998. Epub 2017 Oct 24.

CR-CHUM, Université de Montréal, Montreal, Québec, Canada.

The identification and study of antigen-specific CD4 T cells, both in peripheral blood and in tissues, is key for a broad range of immunological research, including vaccine responses and infectious diseases. Detection of these cells is hampered by both their rarity and their heterogeneity, in particular with regards to cytokine secretion profiles. These factors prevent the identification of the total pool of antigen-specific CD4 T cells by classical methods. We have developed assays for the highly sensitive detection of such cells by measuring the upregulation of surface activation induced markers (AIM). Here, we compare two such assays based on concurrent expression of CD69 plus CD40L (CD154) or expression of OX40 plus CD25, and we develop additional AIM assays based on OX40 plus PD-L1 or 4-1BB. We compare the relative sensitivity of these assays for detection of vaccine and natural infection-induced CD4 T cell responses and show that these assays identify distinct, but overlapping populations of antigen-specific CD4 T cells, a subpopulation of which can also be detected on the basis of cytokine synthesis. Bystander activation had minimal effect on AIM markers. However, some T regulatory cells upregulate CD25 upon antigen stimulation. We therefore validated AIM assays designed to exclude most T regulatory cells, for both human and non-human primate (NHP, Macaca mulatta) studies. Overall, through head-to-head comparisons and methodological improvements, we show that AIM assays represent a sensitive and valuable method for the detection of antigen-specific CD4 T cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0186998PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5655442PMC
November 2017

T cells control the generation of nanomolar-affinity anti-glycan antibodies.

J Clin Invest 2017 Apr 13;127(4):1491-1504. Epub 2017 Mar 13.

Vaccines targeting glycan structures at the surface of pathogenic microbes must overcome the inherent T cell-independent nature of immune responses against glycans. Carbohydrate conjugate vaccines achieve this by coupling bacterial polysaccharides to a carrier protein that recruits heterologous CD4 T cells to help B cell maturation. Yet they most often produce low- to medium-affinity immune responses of limited duration in immunologically fit individuals and disappointing results in the elderly and immunocompromised patients. Here, we hypothesized that these limitations result from suboptimal T cell help. To produce the next generation of more efficacious conjugate vaccines, we have explored a synthetic design aimed at focusing both B cell and T cell recognition to a single short glycan displayed at the surface of a virus-like particle. We tested and established the proof of concept of this approach for 2 serotypes of Streptococcus pneumoniae. In both cases, these vaccines elicited serotype-specific, protective, and long-lasting IgG antibodies of nanomolar affinity against the target glycans in mice. We further identified a requirement for CD4 T cells in the anti-glycan antibody response. Our findings establish the design principles for improved glycan conjugate vaccines. We surmise that the same approach can be used for any microbial glycan of interest.
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http://dx.doi.org/10.1172/JCI91192DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5373877PMC
April 2017

Definition of Human Epitopes Recognized in Tetanus Toxoid and Development of an Assay Strategy to Detect Ex Vivo Tetanus CD4+ T Cell Responses.

PLoS One 2017 12;12(1):e0169086. Epub 2017 Jan 12.

La Jolla Institute for Allergy and Immunology, La Jolla, California, United Ststes of America.

Despite widespread uses of tetanus toxoid (TT) as a vaccine, model antigen and protein carrier, TT epitopes have been poorly characterized. Herein we defined the human CD4+ T cell epitope repertoire by reevaluation of previously described epitopes and evaluation of those derived from prediction of HLA Class II binding. Forty-seven epitopes were identified following in vitro TT stimulation, with 28 epitopes accounting for 90% of the total response. Despite this diverse range of epitopes, individual responses were associated with only a few immunodominant epitopes, with each donor responding on average to 3 epitopes. For the top 14 epitopes, HLA restriction could be inferred based on HLA typing of the responding donors. HLA binding predictions re-identified the vast majority of known epitopes, and identified 24 additional novel epitopes. With these epitopes, we created a TT epitope pool, which allowed us to characterize TT responses directly ex vivo using a cytokine-independent Activation Induced Marker (AIM) assay. These TT responses were highly Th1 or Th2 polarized, which was dependent upon the original priming vaccine, either the cellular DTwP or acellular DTaP formulation. This polarization remained despite the original priming having occurred decades past and a recent booster immunization with a reduced acellular vaccine formulation. While TT responses following booster vaccination were not durably increased in magnitude, they were associated with a relative expansion of CD4+ effector memory T cells.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0169086PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5230748PMC
August 2017

Response to Comment on "A Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal Center T Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood".

J Immunol 2016 10;197(7):2558

La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; Division of Infectious Diseases, University of California, San Diego, La Jolla, CA 92093; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, La Jolla, CA 92037;

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http://dx.doi.org/10.4049/jimmunol.1601321DOI Listing
October 2016

A Cytokine-Independent Approach To Identify Antigen-Specific Human Germinal Center T Follicular Helper Cells and Rare Antigen-Specific CD4+ T Cells in Blood.

J Immunol 2016 08 24;197(3):983-93. Epub 2016 Jun 24.

La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; Division of Infectious Diseases, University of California, San Diego, La Jolla, CA 92093; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, La Jolla, CA 92037; and

Detection of Ag-specific CD4(+) T cells is central to the study of many human infectious diseases, vaccines, and autoimmune diseases. However, such cells are generally rare and heterogeneous in their cytokine profiles. Identification of Ag-specific germinal center (GC) T follicular helper (Tfh) cells by cytokine production has been particularly problematic. The function of a GC Tfh cell is to selectively help adjacent GC B cells via cognate interaction; thus, GC Tfh cells may be stingy cytokine producers, fundamentally different from Th1 or Th17 cells in the quantities of cytokines produced. Conventional identification of Ag-specific cells by intracellular cytokine staining relies on the ability of the CD4(+) T cell to generate substantial amounts of cytokine. To address this problem, we have developed a cytokine-independent activation-induced marker (AIM) methodology to identify Ag-specific GC Tfh cells in human lymphoid tissue. Whereas Group A Streptococcus-specific GC Tfh cells produced minimal detectable cytokines by intracellular cytokine staining, the AIM method identified 85-fold more Ag-specific GC Tfh cells. Intriguingly, these GC Tfh cells consistently expressed programmed death ligand 1 upon activation. AIM also detected non-Tfh cells in lymphoid tissue. As such, we applied AIM for identification of rare Ag-specific CD4(+) T cells in human peripheral blood. Dengue, tuberculosis, and pertussis vaccine-specific CD4(+) T cells were readily detectable by AIM. In summary, cytokine assays missed 98% of Ag-specific human GC Tfh cells, reflecting the biology of these cells, which could instead be sensitively identified by coexpression of TCR-dependent activation markers.
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http://dx.doi.org/10.4049/jimmunol.1600318DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4955771PMC
August 2016

Cytokine-Independent Detection of Antigen-Specific Germinal Center T Follicular Helper Cells in Immunized Nonhuman Primates Using a Live Cell Activation-Induced Marker Technique.

J Immunol 2016 08 22;197(3):994-1002. Epub 2016 Jun 22.

Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037; Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, La Jolla, CA 92037;

A range of current candidate AIDS vaccine regimens are focused on generating protective HIV-neutralizing Ab responses. Many of these efforts rely on the rhesus macaque animal model. Understanding how protective Ab responses develop and how to increase their efficacy are both major knowledge gaps. Germinal centers (GCs) are the engines of Ab affinity maturation. GC T follicular helper (Tfh) CD4 T cells are required for GCs. Studying vaccine-specific GC Tfh cells after protein immunizations has been challenging, as Ag-specific GC Tfh cells are difficult to identify by conventional intracellular cytokine staining. Cytokine production by GC Tfh cells may be intrinsically limited in comparison with other Th effector cells, as the biological role of a GC Tfh cell is to provide help to individual B cells within the GC, rather than secreting large amounts of cytokines bathing a tissue. To test this idea, we developed a cytokine-independent method to identify Ag-specific GC Tfh cells. RNA sequencing was performed using TCR-stimulated GC Tfh cells to identify candidate markers. Validation experiments determined CD25 (IL-2Rα) and OX40 to be highly upregulated activation-induced markers (AIM) on the surface of GC Tfh cells after stimulation. In comparison with intracellular cytokine staining, the AIM assay identified >10-fold more Ag-specific GC Tfh cells in HIV Env protein-immunized macaques (BG505 SOSIP). CD4 T cells in blood were also studied. In summary, AIM demonstrates that Ag-specific GC Tfh cells are intrinsically stingy producers of cytokines, which is likely an essential part of their biological function.
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http://dx.doi.org/10.4049/jimmunol.1600320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4955744PMC
August 2016

Brief Report: Effect of CMV and HIV Transcription on CD57 and PD-1 T-Cell Expression During Suppressive ART.

J Acquir Immune Defic Syndr 2016 06;72(2):133-7

*La Jolla Institute for Allergy and Immunology, La Jolla, CA;†University of California, San Diego, CA;‡Veterans Affairs San Diego Healthcare System, San Diego, CA;§Los Angeles Biomedical Research Institute, Harbor-UCLA Medical Center, Torrance, CA; and‖University of Southern California, Keck School of Medicine, Los Angeles, CA.

HIV-infected men who have sex with men are nearly universally coinfected with cytomegalovirus (CMV). In this study of 45 HIV-infected men who have sex with men virologically suppressed on ART, we found that presence of seminal CMV DNA shedding and higher levels of systemic cellular HIV RNA transcription were both independently associated with increased PD-1 expression on circulating CD4 T cells, but not with higher levels of senescent (CD57) T cells. In addition, greater HIV RNA transcription was associated with lower CD57 expression on CD8 T cells. Although causality cannot be inferred from this retrospective study, these results suggest that asymptomatic CMV replication and residual cellular HIV transcription may contribute to persistent immune dysregulation during suppressive ART.
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http://dx.doi.org/10.1097/QAI.0000000000000936DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4868660PMC
June 2016

Methamphetamine Use in HIV-infected Individuals Affects T-cell Function and Viral Outcome during Suppressive Antiretroviral Therapy.

Sci Rep 2015 Aug 24;5:13179. Epub 2015 Aug 24.

University of California San Diego, La Jolla, CA, United States.

We investigated the associations between methamphetamine (meth) use, immune function, and the dynamics of HIV and cytomegalovirus [CMV] in the blood and genital tract of HIV-infected ART-suppressed subjects. Self-reported meth use was associated with increased CD4(+) and CD8(+) T-cell proliferation (Ki67(+), p < 0.005), CD4(+) T-cell activation (CD45RA(-)CD38(+), p = 0.005) and exhaustion (PD-1(+), p = 0.0004) in blood, compared to non-meth users. Meth use was also associated with a trend towards higher blood HIV DNA levels (p = 0.09) and more frequent shedding of CMV in seminal plasma (p = 0.002). To explore possible mechanisms, we compared ex vivo spontaneous and antigen-specific proliferation in PBMC collected from subjects with and without positive meth detection in urine (Utox+ vs. Utox-). Despite higher levels of spontaneous proliferation, lymphocytes from Utox+ meth users had a significantly lower proliferative capacity after stimulation with a number of pathogens (CMV, candida, mycobacterium, toxoplasma, HIV, p < 0.04 in all cases), compared to Utox- participants. Our findings suggest that meth users have greater proliferation and exhaustion of the immune system. Meth use is also associated with a loss of control of CMV replication, which could be related to loss of immune response to pathogens. Future studies should consider meth use as a potential modulator of T-cell responses.
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http://dx.doi.org/10.1038/srep13179DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4547398PMC
August 2015

Adult intussusception secondary to colorectal cancer in a young man: a case report.

J Emerg Med 2012 Dec 22;43(6):983-6. Epub 2011 Jan 22.

MD-PHD Program, Boston University School of Medicine, Boston, Massachusetts, USA.

Background: The occurrence of adult intussusception from colorectal cancer in a 27-year-old man is quite uncommon.

Objectives: To raise awareness of the incidence of intussusception in adults, to educate others about the protean manifestations and high association with malignancy of the disease, and to provide treatment recommendations.

Case Report: We present a case of a 27-year-old man with a non-contributory family history who presented to the Emergency Department multiple times over a 10-month period with vague abdominal complaints. Clinical symptoms ultimately included a 75-lb weight loss, fatigue, mild right-sided abdominal pain, and anemia. Computed tomography scan of the abdomen revealed right-sided colocolic intussusception with a lead point. The patient underwent a right hemicolectomy with ileocolic anastomosis. Pathologic evaluation and staging revealed a stage IIIB poorly differentiated adenocarcinoma. Molecular analysis was negative for genetic causes.

Conclusion: This case demonstrates how intussusception and possible colorectal cancer must be included in the differential diagnosis even in young adults who have persistent abdominal complaints.
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http://dx.doi.org/10.1016/j.jemermed.2010.11.030DOI Listing
December 2012

Cooperative stimulation of dendritic cells by Cryptococcus neoformans mannoproteins and CpG oligodeoxynucleotides.

PLoS One 2008 Apr 30;3(4):e2046. Epub 2008 Apr 30.

Department of Microbiology and Immunology Training Program, Boston University School of Medicine, Boston, Massachusetts, United States of America.

While mannosylation targets antigens to mannose receptors on dendritic cells (DC), the resultant immune response is suboptimal. We hypothesized that the addition of toll-like receptor (TLR) ligands would enhance the DC response to mannosylated antigens. Cryptococcus neoformans mannoproteins (MP) synergized with CpG-containing oligodeoxynucleotides to stimulate enhanced production of proinflammatory cytokines and chemokines from murine conventional and plasmacytoid DC. Synergistic stimulation required the interaction of mannose residues on MP with the macrophage mannose receptor (MR), CD206. Moreover, synergy with MP was observed with other TLR ligands, including tripalmitoylated lipopeptide (Pam3CSK4), polyinosine-polycytidylic acid (pI:C), and imiquimod. Finally, CpG enhanced MP-specific MHC II-restricted CD4(+) T-cell responses by a mechanism dependent upon DC expression of CD206 and TLR9. These data suggest a rationale for vaccination strategies that combine mannosylated antigens with TLR ligands and imply that immune responses to naturally mannosylated antigens on pathogens may be greatly augmented if TLR and MR are cooperatively stimulated.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0002046PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2297515PMC
April 2008

Role of the mannose receptor in a murine model of Cryptococcus neoformans infection.

Infect Immun 2008 Jun 7;76(6):2362-7. Epub 2008 Apr 7.

Department of Microbiology and Immunology Training Program, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

Cryptococcus neoformans is an encapsulated fungal pathogen with a predilection to infect persons with suppressed T-cell function. Cryptococcal mannoproteins (MP) are highly mannosylated antigens which elicit T-cell responses in infected mice and in convalescent patients. Key to the immunogenicity of MP is its capacity to bind to the conserved mannose receptor (MR), CD206, on dendritic cells (DCs). To test the role of the MR in the immune response to C. neoformans, wild-type and MR knockout (MR KO) mice were compared by using in vivo and ex vivo models of cryptococcosis. Following a pulmonary challenge with C. neoformans, MR KO mice died significantly faster than wild-type mice and had higher lung fungal burdens after 4 weeks of infection. Uptake of MP was similar when DCs obtained from wild-type and MR KO mice were compared. Additionally, MP did not upregulate the maturation markers major histocompatibility complex class II, CD86, and CD40 in either wild-type or MR KO DCs. However, MP stimulated lymphoproliferation in CD4(+) T cells obtained from the peripheral lymph nodes of infected wild-type but not MR KO mice. These studies demonstrate a nonredundant role for the MR in the development of CD4(+) T-cell responses to MP and protection from C. neoformans.
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http://dx.doi.org/10.1128/IAI.00095-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2423054PMC
June 2008

Contribution of glycosylation to T cell responses stimulated by recombinant Cryptococcus neoformans mannoprotein.

J Infect Dis 2007 Sep 20;196(5):796-800. Epub 2007 Jul 20.

University of Massachusetts Medical School, Worcester, MA 01605, USA.

Mannoproteins are major antigens driving T cell responses to the opportunistic fungus Cryptococcus neoformans. To investigate the role played by mannosylation, an immunoreactive cryptococcal mannoprotein was expressed recombinantly in E. coli and Pichia pastoris, resulting in unglycosylated and mannosylated proteins, respectively. The Pichia-derived antigen stimulated stronger major histocompatibility class II-restricted T cell responses. Moreover, responses were potently inhibited if the antigen was chemically deglycosylated or if mannose receptors were blocked with mannans. Thus, mannosylation is critical for optimal T cell responses to a fungal antigen and should be taken into account when vaccines to protect against mycoses are designed.
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http://dx.doi.org/10.1086/520536DOI Listing
September 2007

Prospects for development of vaccines against fungal diseases.

Drug Resist Updat 2006 Jun 3;9(3):105-10. Epub 2006 Jul 3.

Department of Medicine, Boston University School of Medicine, Boston, MA 02118, United States.

Despite recent additions to our antifungal drug armamentarium, success rates for many mycoses remain unacceptably low and antifungal drug therapy is often limited by toxicity, resistance and high cost. To circumvent these difficulties, alternative approaches to prevention and treatment are being developed, including vaccines and passive immunotherapy. Here, we review the progress of current research in this field, discuss some of the potential obstacles to developing and marketing a protective antifungal vaccine, and summarize two clinical trials of monoclonal antibodies as adjunctive treatment of established mycoses. In animal models of fungal infections, protective responses have been elicited with vaccines composed of whole organisms, soluble cell free fractions, purified proteins, glycans and nucleic acids. Methods to boost the immune response to vaccination include the use of adjuvants and antigen-loaded dendritic cells (DCs). A significant challenge to the development of effective vaccines will be to elicit immune responses in immunocompromised individuals who are most at risk for invasive fungal infections.
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http://dx.doi.org/10.1016/j.drup.2006.05.004DOI Listing
June 2006