Foreman, TW;Nelson, CE;Kauffman, KD;Lora, NE;Vinhaes, CL;Dorosky, DE;Sakai, S;Gomez, F;Fleegle, JD;Parham, M;Perera, SR;Lindestam Arlehamn, CS;Sette, A;Tuberculosis Imaging Program, ;Brenchley, JM;Queiroz, ATL;Andrade, BB;Kabat, J;Via, LE;Barber, DL;
PMID: 35649361 | DOI: 10.1016/j.celrep.2022.110896
HIV/Mycobacterium tuberculosis (Mtb) co-infected individuals have an increased risk of tuberculosis prior to loss of peripheral CD4 T cells, raising the possibility that HIV co-infection leads to CD4 T cell depletion in lung tissue before it is evident in blood. Here, we use rhesus macaques to study the early effects of simian immunodeficiency virus (SIV) co-infection on pulmonary granulomas. Two weeks after SIV inoculation of Mtb-infected macaques, Mtb-specific CD4 T cells are dramatically depleted from granulomas, before CD4 T cell loss in blood, airways, and lymph nodes, or increases in bacterial loads or radiographic evidence of disease. Spatially, CD4 T cells are preferentially depleted from the granuloma core and cuff relative to B cell-rich regions. Moreover, live imaging of granuloma explants show that intralesional CD4 T cell motility is reduced after SIV co-infection. Thus, granuloma CD4 T cells may be decimated before many co-infected individuals experience the first symptoms of acute HIV infection.
The Journal of clinical investigation
Duhen, R;Fesneau, O;Samson, KA;Frye, AK;Beymer, M;Rajamanickam, V;Ross, D;Tran, E;Bernard, B;Weinberg, AD;Duhen, T;
PMID: 35439168 | DOI: 10.1172/JCI156821
CD4 T helper (Th) cells play a key role in orchestrating immune responses, but the identity of the CD4 Th cells involved in the anti-tumor immune response remains to be defined. We analyzed the immune cell infiltrates of head and neck squamous cell carcinoma and colorectal cancers and identified a subset of CD4 Th cells distinct from FOXP3+ regulatory T cells that co-express PD-1 and ICOS. These tumor-infiltrating CD4 Th cells (CD4 Th TIL) have a tissue-resident memory phenotype, are present in MHC class II-rich areas and proliferate in the tumor suggesting local antigen recognition. The T-cell receptor repertoire of the PD-1+ICOS+ CD4 Th TIL is oligoclonal, with T-cell clones expanded in the tumor, but present at low frequencies in the periphery. Finally, these PD-1+ICOS+ CD4 Th TIL were shown to recognize both tumor-associated antigens and tumor-specific neoantigens. Our findings provide an approach for isolating tumor-reactive CD4 Th TIL directly ex vivo that will help define their role in the anti-tumor immune response and potentially improve future adoptive T-cell therapy approaches.
Vinton CL, Ortiz AM, Calantone N, Mudd JC, Deleage C, Morcock DR, Whitted S, Estes JD, Hirsch VM, Brenchley JM.
PMID: 28438898 | DOI: 10.4049/jimmunol.1700136
African green monkeys (AGMs) are a natural host of SIV that do not develop simian AIDS. Adult AGMs naturally have low numbers of CD4+T cells and a large population of MHC class II-restricted CD8αα T cells that are generated through CD4 downregulation in CD4+ T cells. In this article, we study the functional profiles and SIV infection status in vivo of CD4+ T cells, CD8αα T cells, and CD8αβ T cells in lymph nodes, peripheral blood, and bronchoalveolar lavage fluid of AGMs and rhesus macaques (in which CD4 downregulation is not observed). We show that, although CD8αα T cells in AGMs maintain functions associated with CD4+ T cells (including Th follicular functionality in lymphoid tissues and Th2 responses in bronchoalveolar lavage fluid), they also accumulate functions normally attributed to canonical CD8+ T cells. These hyperfunctional CD8αα T cells are found to circulate peripherally, as well as reside within the lymphoid tissue. Due to their unique combination of CD4 and CD8 T cell effector functions, these CD4- CD8αα T cells are likely able to serve as an immunophenotype capable of Th1, follicular Th, and CTL functionalities, yet they are unable to be infected by SIV. These data demonstrate the ambiguity of CD4/CD8 expression in dictating the functional capacities of T cells and suggest that accumulation of hyperfunctional CD8αα T cells in AGMs may lead to tissue-specific antiviral immune responses in lymphoid follicles that limit SIV replication in this particular anatomical niche.
Science translational medicine
Selvanesan, BC;Chandra, D;Quispe-Tintaya, W;Jahangir, A;Patel, A;Meena, K;Alves Da Silva, RA;Friedman, M;Gabor, L;Khouri, O;Libutti, SK;Yuan, Z;Li, J;Siddiqui, S;Beck, A;Tesfa, L;Koba, W;Chuy, J;McAuliffe, JC;Jafari, R;Entenberg, D;Wang, Y;Condeelis, J;DesMarais, V;Balachandran, V;Zhang, X;Lin, K;Gravekamp, C;
PMID: 35320003 | DOI: 10.1126/scitranslmed.abc1600
Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic disease. Tumors are poorly immunogenic and immunosuppressive, preventing T cell activation in the tumor microenvironment. Here, we present a microbial-based immunotherapeutic treatment for selective delivery of an immunogenic tetanus toxoid protein (TT856-1313) into PDAC tumor cells by attenuated Listeria monocytogenes. This treatment reactivated preexisting TT-specific memory T cells to kill infected tumor cells in mice. Treatment of KrasG12D,p53R172H, Pdx1-Cre (KPC) mice with Listeria-TT resulted in TT accumulation inside tumor cells, attraction of TT-specific memory CD4 T cells to the tumor microenvironment, and production of perforin and granzyme B in tumors. Low doses of gemcitabine (GEM) increased immune effects of Listeria-TT, turning immunologically cold into hot tumors in mice. In vivo depletion of T cells from Listeria-TT + GEM-treated mice demonstrated a CD4 T cell-mediated reduction in tumor burden. CD4 T cells from TT-vaccinated mice were able to kill TT-expressing Panc-02 tumor cells in vitro. In addition, peritumoral lymph node-like structures were observed in close contact with pancreatic tumors in KPC mice treated with Listeria-TT or Listeria-TT + GEM. These structures displayed CD4 and CD8 T cells producing perforin and granzyme B. Whereas CD4 T cells efficiently infiltrated the KPC tumors, CD8 T cells did not. Listeria-TT + GEM treatment of KPC mice with advanced PDAC reduced tumor burden by 80% and metastases by 87% after treatment and increased survival by 40% compared to nontreated mice. These results suggest that Listeria-delivered recall antigens could be an alternative to neoantigen-mediated cancer immunotherapy.
Plasmacytoid dendritic cells have divergent effects on HIV infection of initial target cells and induce a pro-retention phenotype
Tong, O;Duette, G;O'Neil, TR;Royle, CM;Rana, H;Johnson, B;Popovic, N;Dervish, S;Brouwer, MAE;Baharlou, H;Patrick, E;Ctercteko, G;Palmer, S;Lee, E;Hunter, E;Harman, AN;Cunningham, AL;Nasr, N;
PMID: 33872331 | DOI: 10.1371/journal.ppat.1009522
Although HIV infection inhibits interferon responses in its target cells in vitro, interferon signatures can be detected in vivo soon after sexual transmission, mainly attributed to plasmacytoid dendritic cells (pDCs). In this study, we examined the physiological contributions of pDCs to early HIV acquisition using coculture models of pDCs with myeloid DCs, macrophages and the resting central, transitional and effector memory CD4 T cell subsets. pDCs impacted infection in a cell-specific manner. In myeloid cells, HIV infection was decreased via antiviral effects, cell maturation and downregulation of CCR5 expression. In contrast, in resting memory CD4 T cells, pDCs induced a subset-specific increase in intracellular HIV p24 protein expression without any activation or increase in CCR5 expression, as measured by flow cytometry. This increase was due to reactivation rather than enhanced viral spread, as blocking HIV entry via CCR5 did not alter the increased intracellular p24 expression. Furthermore, the load and proportion of cells expressing HIV DNA were restricted in the presence of pDCs while reverse transcriptase and p24 ELISA assays showed no increase in particle associated reverse transcriptase or extracellular p24 production. In addition, pDCs also markedly induced the expression of CD69 on infected CD4 T cells and other markers of CD4 T cell tissue retention. These phenotypic changes showed marked parallels with resident memory CD4 T cells isolated from anogenital tissue using enzymatic digestion. Production of IFNα by pDCs was the main driving factor for all these results. Thus, pDCs may reduce HIV spread during initial mucosal acquisition by inhibiting replication in myeloid cells while reactivating latent virus in resting memory CD4 T cells and retaining them for immune clearance.
Hasegawa, T;Oka, T;Son, H;Azin, M;Eisenhaure, T;Lieb, D;Hacohen, N;Demehri, S;
| DOI: 10.2139/ssrn.4102631
Senescent cell accumulation has been implicated in the pathogenesis of aging-associated diseases including cancer. The mechanism that prevents the accumulation of senescent cells in aging human organs is unclear. Here, we demonstrate that a commensal virus-immune axis controls the senescent fibroblast accumulation in the human skin. Senescent fibroblasts increased in old compared with young skin. However, they did not increase with advancing age in elderly. Increased CXCL9 and cytotoxic CD4+ T cell (CD4 CTL) recruitment were significantly associated with reduced senescent fibroblasts in the old skin. Senescent fibroblasts expressed human leukocyte antigen class II (HLA-II) and human cytomegalovirus glycoprotein B (HCMV-gB), becoming direct CD4 CTL targets. Skin-resident CD4 CTL eliminated HCMV-gB+ senescent fibroblasts in an HLA-II-dependent manner and HCMV-gB activated CD4 CTL from the human skin. Collectively, our findings demonstrate HCMV reactivation in senescent cells, which can be directly eliminated by CD4 CTL through the recognition of the HCMV-gB antigen.
Hasegawa, T;Oka, T;Son, HG;Oliver-García, VS;Azin, M;Eisenhaure, TM;Lieb, DJ;Hacohen, N;Demehri, S;
PMID: 37001502 | DOI: 10.1016/j.cell.2023.02.033
Senescent cell accumulation has been implicated in the pathogenesis of aging-associated diseases, including cancer. The mechanism that prevents the accumulation of senescent cells in aging human organs is unclear. Here, we demonstrate that a virus-immune axis controls the senescent fibroblast accumulation in the human skin. Senescent fibroblasts increased in old skin compared with young skin. However, they did not increase with advancing age in the elderly. Increased CXCL9 and cytotoxic CD4+ T cells (CD4 CTLs) recruitment were significantly associated with reduced senescent fibroblasts in the old skin. Senescent fibroblasts expressed human leukocyte antigen class II (HLA-II) and human cytomegalovirus glycoprotein B (HCMV-gB), becoming direct CD4 CTL targets. Skin-resident CD4 CTLs eliminated HCMV-gB+ senescent fibroblasts in an HLA-II-dependent manner, and HCMV-gB activated CD4 CTLs from the human skin. Collectively, our findings demonstrate HCMV reactivation in senescent cells, which CD4 CTLs can directly eliminate through the recognition of the HCMV-gB antigen.
HIV DNA reservoir and elevated PD-1 expression of CD4 T-cell subsets particularly persist in the terminal ileum of HIV-positive patients despite cART
Horn, C;Augustin, M;Ercanoglu, MS;Heger, E;Knops, E;Bondet, V;Duffy, D;Chon, SH;Nierhoff, D;Oette, M;Schäfer, H;Vivaldi, C;Held, K;Anderson, J;Geldmacher, C;Suárez, I;Rybniker, J;Klein, F;Fätkenheuer, G;Müller-Trutwin, M;Lehmann, C;
PMID: 33421299 | DOI: 10.1111/hiv.13031
Despite its importance as an HIV anatomic sanctuary, little is known about the characteristics of the HIV reservoir in the terminal ileum (TI). In blood, the immune checkpoint inhibitor programmed-death-1 (PD-1) has been linked to the HIV reservoir and T-cell immune dysfunction. We thus evaluated PD-1 expression and cell-associated HIV DNA in memory CD4 T-cell subsets from TI, peripheral blood (PB) and rectum (RE) of untreated and treated HIV-positive patients to identify associations between PD-1 and HIV reservoir in other sites. Using mononuclear cells from PB, TI and RE of untreated HIV-positive (N = 6), treated (n = 18) HIV-positive and uninfected individuals (n = 16), we identified and sorted distinct memory CD4 T-cell subsets by flow cytometry, quantified their cell-associated HIV DNA using quantitative PCR and assessed PD-1 expression levels using geometric mean fluorescence intensity. Combined HIV-1 RNA in situ hybridization and immunohistochemistry was performed on ileal biopsy sections. Combined antiretroviral therapy (cART)-treated patients with undetectable HIV RNA and significantly lower levels of HIV DNA in PB showed particularly high PD-1 expression in PB and TI, and high HIV DNA levels in TI, irrespective of clinical characteristics. By contrast, in treatment-naïve patients HIV DNA levels in memory CD4 T-cell subsets were high in PB and TI. Elevated PD-1 expression on memory CD4 T-cells in PB and TI despite treatment points to continuous immune dysfunction and underlines the importance of evaluating immunotherapy in reversing HIV latency and T-cell reconstitution. As HIV DNA particularly persists in TI despite cART, investigating samples from TI is crucial in understanding HIV immunopathogenesis.
Cytometry A. 2014 Aug 13.
Van Hoof D, Lomas W, Hanley MB, Park E.
PMID: 25123411 | DOI: 10.1002/cyto.a.22521.
The application of fluorescently-labeled antibodies for flow cytometric identification and characterization of specific cell types within heterogeneous populations by their protein expression profile is well established. While detection of proteins is informative, concomitant transcript analysis in the same cells would provide a more complete and comprehensive view of intracellular signaling events. We recently reported on the efficient detection of RNA in suspension cells for flow cytometric analysis. The improved RNA flow cytometry procedure described here allows for the specific labeling of multiple RNA species, and is compatible with antibody-based targeting of extracellular and intracellular antigens for multiplexing purposes. To show proof of concept, human peripheral blood mononuclear cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin for a maximum of 5 h, during which their CD4 and interferon-gamma (IFN-γ) transcript and protein levels were monitored. Substantial and increasing numbers of IFN-γ mRNA+ cells were detected within 30 min after initiation of induction, while IFN-γ protein+ cells could only be discerned at 1 h and beyond. Surprisingly, resting lymphocytes contained less CD4 mRNA but more of the protein per cell compared with monocytes, revealing a difference in the relationship of transcript and protein levels in these two cell types. We additionally applied monensin, which is commonly used to block cytokine secretion, and found that IFN-γ mRNA can still be analyzed consistently using the improved RNA flow cytometry staining method. Notably, a subset of IFN-γ mRNA- /protein+ cells that were not observed in the absence of monensin became apparent at the 5-h mark. This subset probably represents cells that have accumulated IFN-γ protein, but no longer transcribe mRNA. Collectively, the results described here exemplify how the improved RNA flow cytometry labeling procedure can be applied to simultaneously assess mRNA and protein dynamics to gain insight into the regulation of gene transcription and translation in individual cells. © 2014 International Society for Advancement of Cytometry.
Kovarova, M;Wessel, SE;Johnson, CE;Anderson, SV;Cottrell, ML;Sykes, C;Cohen, MS;Garcia, JV;
PMID: 37306625 | DOI: 10.1128/mbio.02224-22
Sexually transmitted HIV infections in heterosexual men are acquired through the penis. Low adherence to condom usage and the fact that 40% of circumcised men are not protected indicate the need for additional prevention strategies. Here, we describe a new approach to evaluate the prevention of penile HIV transmission. We demonstrated that the entire male genital tract (MGT) of bone marrow/liver/thymus (BLT) humanized mice is repopulated with human T and myeloid cells. The majority of the human T cells in the MGT express CD4 and CCR5. Direct penile exposure to HIV leads to systemic infection including all tissues of the MGT. HIV replication throughout the MGT was reduced 100-1,000-fold by treatment with 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), resulting in the restoration of CD4+ T cell levels. Importantly, systemic preexposure prophylaxis with EFdA effectively protects from penile HIV acquisition.IMPORTANCEOver 84.2 million people have been infected by the human immunodeficiency virus type 1 (HIV-1) during the past 40 years, most through sexual transmission. Men comprise approximately half of the HIV-infected population worldwide. Sexually transmitted HIV infections in exclusively heterosexual men are acquired through the penis. However, direct evaluation of HIV infection throughout the human male genital tract (MGT) is not possible. Here, we developed a new in vivo model that permits, for the first time, the detail analysis of HIV infection. Using BLT humanized mice, we showed that productive HIV infection occurs throughout the entire MGT and induces a dramatic reduction in human CD4 T cells compromising immune responses in this organ. Antiretroviral treatment with novel drug EFdA suppresses HIV replication in all tissues of the MGT, restores normal levels of CD4 T cells and is highly efficient at preventing penile transmission.
In Situ Characterization of Human Lymphoid Tissue Immune Cells by Multispectral Confocal Imaging and Quantitative Image Analysis; Implications for HIV Reservoir Characterization
Moysi, E;Del Rio Estrada, PM;Torres-Ruiz, F;Reyes-Terán, G;Koup, RA;Petrovas, C;
PMID: 34177929 | DOI: 10.3389/fimmu.2021.683396
CD4 T cells are key mediators of adaptive immune responses during infection and vaccination. Within secondary lymphoid organs, helper CD4 T cells, particularly those residing in germinal centers known as follicular helper T cells (Tfh), provide critical help to B-cells to promote their survival, isotype switching and selection of high affinity memory B-cells. On the other hand, the important role of Tfh cells for the maintenance of HIV reservoir is well documented. Thus, interrogating and better understanding the tissue specific micro-environment and immune subsets that contribute to optimal Tfh cell differentiation and function is important for designing successful prevention and cure strategies. Here, we describe the development and optimization of eight multispectral confocal microscopy immunofluorescence panels designed for in depth characterization and immune-profiling of relevant immune cells in formalin-fixed paraffin-embedded human lymphoid tissue samples. We provide a comprehensive library of antibodies to use for the characterization of CD4+ T-cells -including Tfh and regulatory T-cells- as well as CD8 T-cells, B-cells, macrophages and dendritic cells and discuss how the resulting multispectral confocal datasets can be quantitatively dissected using the HistoCytometry pipeline to collect information about relative frequencies and immune cell spatial distributions. Cells harboring actively transcribed virus are analyzed using an in-situ hybridization assay for the characterization of HIV mRNA positive cells in combination with additional protein markers (multispectral RNAscope). The application of this methodology to lymphoid tissues offers a means to interrogate multiple relevant immune cell targets simultaneously at increased resolution in a reproducible manner to guide CD4 T-cell studies in infection and vaccination.
McGary CS, Deleage C, Harper J, Micci L, Ribeiro SP, Paganini S, Kuri-Cervantes L, Benne C, Ryan ES, Balderas R, Jean S, Easley K, Marconi V, Silvestri G, Estes JD, Sekaly RP, Paiardini M.
PMID: 29045906 | DOI: 10.1016/j.immuni.2017.09.018
Antiretroviral therapy (ART) suppresses viral replication in HIV-infected individuals but does not eliminate the reservoir of latently infected cells. Recent work identified PD-1+ follicular helper T (Tfh) cells as an important cellular compartment for viral persistence. Here, using ART-treated, SIV-infected rhesus macaques, we show that CTLA-4+PD-1- memory CD4+ T cells, which share phenotypic markers with regulatory T cells, were enriched in SIV DNA in blood, lymph nodes (LN), spleen, and gut, and contained replication-competent and infectious virus. In contrast to PD-1+ Tfh cells, SIV-enriched CTLA-4+PD-1- CD4+ T cells were found outside the B cell follicle of the LN, predicted the size of the persistent viral reservoir during ART, and significantly increased their contribution to the SIV reservoir with prolonged ART-mediated viral suppression. We have shown that CTLA-4+PD-1- memory CD4+ T cells are a previously unrecognized component of the SIV and HIV reservoir that should be therapeutically targeted for a functional HIV-1 cure.
