Generation of Combined CD8+ and CD4+ T Cell Lines with High Specificity for Adenovirus Hexon Epitopes for Adoptive Immunotherapy after Allogeneic Stem Cell Transplantation.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2225-2225
Author(s):  
Maarten L. Zandvliet ◽  
J.H. Frederik Falkenburg ◽  
Louise A. Veltrop-Duits ◽  
Marco W. Schilham ◽  
Roelof Willemze ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Peptide Pool ◽  
Cd4 T Cell ◽  
Healthy Individuals ◽  
Hexon Protein ◽  
T Cell Lines

Abstract Human Adenovirus (HAdV) can cause serious morbidity in immunocompromised patients, in particular in pediatric recipients of allogeneic stem cell transplantation (alloSCT). Progression to disseminated adenoviral disease is associated with a high mortality, despite treatment with antiviral agents such as ribavirin and cidofovir. It has been demonstrated that reconstitution of HAdV-specific T cells is essential to control adenoviral infection after alloSCT. Adoptive transfer of donor-derived HAdV-specific T cells may therefore be a strategy to provide long-term protection from HAdV. In healthy individuals, T cells directed against HAdV are only detected at low frequencies and are predominantly directed to the HAdV hexon protein. Only recently, a number of immunodominant CD8+ and CD4+ epitopes of HAdV hexon have been defined. Since these epitopes are largely conserved between the different HAdV subgroups, T cells specific for these immunodominant epitopes may provide protection from a wide range of adenoviral serotypes. The aim of this study was to develop a method for the generation of combined CD8+ and CD4+ T cell lines with high and well defined specificity for the HAdV hexon protein. We first analyzed the frequencies of HAdV hexon-specific CD8+ and CD4+ T cells in healthy individuals using sensitive measurement by peptide-MHC tetramers, and intracellular cytokine staining combined with CD154 or peptide-MHC tetramer staining, after stimulation with defined MHC class I peptides, 30-mer peptides containing class II epitopes, or a HAdV hexon protein-spanning pool of overlapping 15-mer peptides (Miltenyi Biotec, Germany). We demonstrated that the frequencies of HAdV hexon-specific T cells were very low in most healthy individuals tested. HAdV hexon-specific CD8+ T cells were detectable in only 3/15 individuals (range 0.16–0.43% of CD8+ T cells), and hexon-specific CD4+ T cells were detected in all individuals with a median of 0.07% (range 0.004–0.38% of CD4+ T cells). The highest frequencies were found after stimulation with the hexon protein-spanning 15-mer peptide pool, indicating activation of both known and unknown epitopes. Kinetic analysis showed highest levels of IFNg production after 4–8 hours of stimulation for HAdV-specific CD8+ T cells, and after 4–48 hours of stimulation for HAdV-specific CD4+ T cells. The phenotype of these HAdV hexon-specific T cells corresponded to an early memory phenotype, CD27+, CD28+, CD62L+, CD45RO+. Despite these low or undetectable frequencies of HAdV-specific T cells, IFNg-based enrichment 4 hours after activation with the HAdV hexon protein-spanning peptide pool resulted in efficient isolation of CD8+ and CD4+ T cells recognizing both known and unknown HAdV hexon epitopes. Following a short culture period of 7 days, the T cell lines consisted of 49–80% CD8+ T cells and 13–15% CD4+ T cells. Restimulation by autologous EBV-LCL loaded with HAdV hexon peptide pool followed by intracellular IFNg staining showed that the frequency of HAdV-specific T cells was increased to 65–95% of CD8+ T cells, and 38–72% of CD4+ T cells. The frequency of HAdV-tetramer-positive cells was increased to 32–76% of CD8+ T cells, indicating that part of HAdV-specific CD8+ T cells recognized known epitopes. After 14 days, the frequency of HAdV-specific T cells had further increased to 89–94% of CD8+ T cells and 61–91% of CD4+ T cells. Starting with only 25x106 donor peripheral blood mononuclear cells, this strategy yielded T cell lines containing 1.3–2.7x106 HAdV-specific combined CD8+ and CD4+ T cells in 14 days. We conclude that we developed a GMP-grade method for the fast generation of highly HAdV-specific CD8+ and CD4+ T cell lines from all healthy donors tested, irrespective of HLA-restriction, for the treatment HAdV infection after alloSCT, with very limited risk of graft-versus-host disease.

Adenovirus Specific T-Cell Lines Devoid of Alloreactivity Against Haploidentical Recipients Can Be Obtained Using a Set of Adenovirus Hexon Peptides.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3273-3273
Author(s):  
Patrizia Comoli ◽  
Marco W. Schilham ◽  
Sabrina Basso ◽  
Tamara van Vreeswijk ◽  
Rita Maccario ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Peptide Pool ◽  
Specific Lysis ◽  
Hematopoietic Stem ◽  
Hexon Protein ◽  
T Cell Lines

Abstract Human Adenovirus (HAdV) infection/reactivation may cause life-threatening complications in recipients of hematopoietic stem cell transplantation (HSCT), the highest risk being observed in pediatric recipients of a T-cell depleted allograft from haploidentical family donor. The effectiveness of pharmacological therapy for HAdV infection is still suboptimal. It has been recently demonstrated that cell therapy may offer a unique opportunity to restore antiviral immune surveillance, leading to clearance of infection and prevention/treatment of disease. However, infusion of HAdV-specific T-cells in the haplo-HSCT cohort poses the concern that GVHD may ensue as a consequence of T-cell transfer. We have conducted scale-up experiments to validate a method of in vitro culture to expand T-cells specific for HAdV, based on stimulation of donor peripheral blood mononuclear cells (PBMC) with a pool of 5 30-mer peptides derived from HAdV5 hexon protein, for use in recipients of haplo-HSCT (Veltrop-Duits et al, Eur J Immunol36, p2410; 2006). A total of 20 T-cell lines were generated, starting from a median of 20 × 106 donor PBMC, that yielded a median of 80 × 106 cells. Most of the cell lines obtained included a majority of CD4+ T-lymphocytes, with a lower % CD8+ cells (median and range: 78, 19–94 and 18, 5–58, respectively) but 5/20 lines contained a high number of CD8+ T cells (ranging between 43% and 58%), which were CD56+ and/or TCRγδ+, and in 1 case also 44% NK cells. Eighteen of the 20 T-cell lines were HAdV-specific, since they showed a median proliferation to the HAdV hexon peptide pool and inactivated HAdV of 14615 (95%CI 8924–31532) and 11103 (95%CI 8805–30174) cpm/105 cells after subtraction of background (responders+irradiated autologous PBMC), respectively. HAdV-specific lysis >10% at a 2:1 effector to target (E:T) ratio was observed in 50% of the T-cell lines. The 2 non-specific, as well as the 3 T-cell lines with lower specific activity, included >40% CD8+ T-cells. Production of IFNγ in an ELIspot assay to HAdV hexon peptide pool above 40 SFU/105 cells was observed in 9 out of 13 tested T-cell lines. Evaluation of specific response to hexon peptides in showed a majority of responses to II42 (80%), with 50–60% responses to II50, II57, II61, and II64. Only 2 out of the 20 T-cell lines tested were prevalently alloreactive against the recipient. Of the 18 HAdV-specific lines, 1 showed higher proliferation to patient PBMC than to HAdV (13518 vs 11717 mean cpm), and would have thus been discarded as unsuitable for in vivo use, while the other 17 showed no alloreactivity (14) or alloreactivity between 10 and 23% of specific proliferation (3). None of these 18 T-cell lines showed lysis >5% against recipient PHA blasts in the cytotoxicity assay. Our data show that PBMC stimulation with HAdV hexon protein-derived 30-mer peptides is able to reproducibly induce the generation of HAdV-specific CD4+ T-cell lines with efficient in vitro antiviral response in most HLA-mismatched HSCT donors. The majority of these T-cell lines show low/undetectable alloreactivity against recipient targets, and could therefore be safely employed for adoptive treatment of HAdV complications developing after HSCT from a HLA-haploidentical donor.

Universal CD137 Expression upon Activation Allows Efficient Isolation of a Broad Repertoire of Virus-Specific CD8+ and CD4+ T Cells for Adoptive Immunotherapy.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2222-2222
Author(s):  
Maarten L. Zandvliet ◽  
J.H. Frederik Falkenburg ◽  
Inge Jedema ◽  
Roelof Willemze ◽  
Henk-Jan Guchelaar ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cd8 T Cell ◽  
Cell Populations ◽  
T Cell Lines ◽  
Kinetics Of ◽  
Ifng Production

Abstract Reactivation of adenovirus (ADV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) can cause serious morbidity and mortality during the prolonged period of immune deficiency following allogeneic stem cell transplantation. It has been shown that adoptive transfer of donor-derived virus-specific T cells can be a successful strategy to control viral reactivation. To provide safe and effective anti-viral immunotherapy, we aimed to generate combined CD8+ and CD4+ T cell lines with high specificity for a broad range of viral epitopes. Isolation by the IFNg capture assay of virus-specific T cells that produce IFNg upon activation allows the generation of highly specific T cell lines without the need for extensive culture. However, it has been recently shown that specific upregulation of the co-stimulatory molecule CD137 upon antigen-specific activation of CD8+ and CD4+ T cells can also be used for isolation. We therefore analyzed IFNg production and CD137 expression by CD8+ and CD4+ T cells upon incubation of peripheral blood mononuclear cells (PBMC) from seropositive donors with peptides corresponding to 17 defined MHC class I restricted minimal epitopes from 10 different ADV, CMV, EBV and influenza (FLU) proteins, and 15-mer or 30-mer peptides containing MHC class II restricted epitopes from CMV pp65 or ADV hexon. Using tetramer and intracellular IFNg staining we first determined the fraction of CD8+ T cells that produced IFNg upon activation with the minimal epitopes. Specific IFNg production was observed for 58–100% of tetramer+ CD8+ T cells specific for CMV pp65 (n=6), and 83% for FLU (n=1), but only 18–58% for CMV pp50 (n=3) or IE-1 (n=3), 4–91% for EBV latent (n=3) and lytic (n=3) epitopes, and 41–63% for ADV hexon (n=2). In contrast to the variation in the fraction of IFNg-producing cells, we observed homogeneous upregulation of CD137 by the virus-specific tetramer+ T cell populations upon activation. In 2 cases where no CD137 expression by tetramer+ T cells could be detected, no IFNg production was observed either. These data suggest that the majority of CD8+ T cells specific for CMV pp65 or FLU can be isolated on basis of IFNg production, but only part of CD8+ T cell populations specific for other viral proteins, while complete virus-specific CD8+ T cell populations may be isolated on basis of CD137 expression. Activation of CD4+ T cells specific for CMV pp65 or ADV hexon with 15-mer or 30-mer peptides induced both specific IFNg production and CD137 expression. To investigate whether multiple virus-specific T cell populations could be isolated simultaneously, we next determined the kinetics of IFNg production after activation with defined MHC class I epitopes or peptides containing MHC class II epitopes. CMV- and EBV-specific CD8+ T cells and CMV-specific CD4+ T cells showed a rapid induction of IFNg production, which peaked after 4 hours and decreased thereafter. In contrast, ADV- and FLU-specific CD8+ T cells and ADV-specific CD4+ T cells, predominantly having a more early differentiation phenotype (CD27+CD28+) compared to CMV- and EBV-specific T cells, showed peak IFNg production after 8 hours that continued for more than 48 hours. This difference in phenotype and IFNg kinetics may suggest that the persistent and frequent presentation of CMV and EBV epitopes in vivo, in contrast to an intermittent exposure to ADV and FLU epitopes, drives differentiation and shapes the kinetics of the IFNg response of specific T cells. Kinetic analysis of CD137 expression showed uniform upregulation by virus-specific CD8+ T cell populations from day 1 to day 4 after activation, which peaked at day 2, suggesting that this may be the optimal time point for CD137-based isolation. In a limited number of experiments, virus-specific CD8+ and CD4+ T cells could be isolated based on CD137 expression within the same timeframe. These data indicate that virus-specific T cell populations can be more efficiently isolated at one time point on basis of CD137 expression than on basis of IFNg production, due to differences in IFNg kinetics. In conclusion, this study shows that T cell lines generated by CD137 isolation may comprise a significant number of virus-specific T cells which do not produce IFNg, but may have other effector functions. Furthermore, CD137-based enrichment may be more robust and allows the efficient simultaneous isolation of multiple virus-specific T cell populations due to uniform kinetics of CD137 expression.

A Novel Strategy for Generation of Human Tumor-Specific T Cell Clones for Adoptive Transfer.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3713-3713
Author(s):  
Seung-Tae Lee ◽  
Shujuan Liu ◽  
Pariya Sukhumalchandra ◽  
Jeffrey Molldrem ◽  
Patrick Hwu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tumor Cells ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Autologous Tumor ◽  
T Cell Clones ◽  
Cell Clones ◽  
T Cell Lines

Abstract Adoptive T-cell therapy using donor lymphocyte infusions is a promising approach for treating hematological malignancies. But, efficacy is limited by the induction of graft-versus-host disease. Transfer of tumor-specific T-cell clones could enhance the graft-versus-tumor effect and eliminate graft-versus-host disease. However, isolating antigen-specific T-cell clones by the traditional limiting dilution approach is a time-consuming and laborious process. Here, we describe a novel strategy for rapidly cloning tumor-specific T cells. Lymphoma-specific T-cell lines were generated from two follicular lymphoma patients by repeated in vitro stimulation of lymphocytes isolated from tumor or blood with autologous soluble CD40 ligand-activated tumor cells. After four in vitro stimulations at 10-day intervals in the presence of IL-2 and IL-15, T-cell lines were found to be predominantly CD4+ T cells and produced significant amounts of TNF-a, GM-CSF, and IFN-γ in response to autologous tumor cells. The tumor reactivity was MHC class II restricted suggesting that it was mediated by CD4+ T cells. Staining with a TCR Vb antibody panel, a set of monoclonal antibodies against 24 human TCR Vb families, revealed that certain Vb families were overrepresented in each CD4+ T-cell line. In patient 1, 51% of CD4+ T cells were Vb1 positive, and in patient 2, 27% of CD4+ T cells were Vb8 positive. To clone lymphoma-specific T cells, CD4+ T-cell lines were labeled with CFSE and stimulated with autologous tumor cells. After 9 days of in vitro expansion in the presence of IL-2 and IL-15, CD4+ T-cell lines were stained with an anti-human CD4-APC monoclonal antibody and an anti-human TCR Vb-PE monoclonal antibody for each CD4+ T-cell line. Proliferating Vb1 cells from patient 1 and Vb8 cells from patient 2 were identified by their reduction in CFSE staining, and CD4+TCRV b +CFSEdim cells were sorted by flow cytometer. Monoclonality of the sorted cells was confirmed by PCR using a panel of optimized primers specific for 24 TCR Vb families, by TCR Vb spectratype analysis, and finally, by sequencing the TCR Vb gene used by each T-cell clone. Sorted tumor-specific T-cell clones could be expanded to large numbers using a 14-day rapid expansion protocol with allofeeder PBMCs, and confirmed to retain specificity against autologous tumor cells in a cytokine induction assay. This approach was also successfully used to isolate melanoma-specific CD8+ T-cell clones from two patients. We conclude that this approach is highly reproducible, rapid, and efficient for generating antigen-specific T-cell clones for adoptive T-cell therapy against human malignancies in the autologous or allogeneic setting.

scholarly journals Reduced immune-regulatory molecule expression on human colonic memory CD4 T cells in older adults

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Stephanie M. Dillon ◽  
Tezha A. Thompson ◽  
Allison J. Christians ◽  
Martin D. McCarter ◽  
Cara C. Wilson
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Older Persons ◽  
Age Groups ◽  
T Cell Immunity ◽  
Cd4 T Cell ◽  
Cell Immunity ◽  
Memory Cd4 T Cells

Abstract Background The etiology of the low-level chronic inflammatory state associated with aging is likely multifactorial, but a number of animal and human studies have implicated a functional decline of the gastrointestinal immune system as a potential driver. Gut tissue-resident memory T cells play critical roles in mediating protective immunity and in maintaining gut homeostasis, yet few studies have investigated the effect of aging on human gut T cell immunity. To determine if aging impacted CD4 T cell immunity in the human large intestine, we utilized multi-color flow cytometry to measure colonic lamina propria (LP) CD4 T cell frequencies and immune-modulatory marker expression in younger (mean ± SEM: 38 ± 1.5 yrs) and older (77 ± 1.6 yrs) adults. To determine cellular specificity, we evaluated colon LP CD8 T cell frequency and phenotype in the same donors. To probe tissue specificity, we evaluated the same panel of markers in peripheral blood (PB) CD4 T cells in a separate cohort of similarly aged persons. Results Frequencies of colonic CD4 T cells as a fraction of total LP mononuclear cells were higher in older persons whereas absolute numbers of colonic LP CD4 T cells per gram of tissue were similar in both age groups. LP CD4 T cells from older versus younger persons exhibited reduced CTLA-4, PD-1 and Ki67 expression. Levels of Bcl-2, CD57, CD25 and percentages of activated CD38+HLA-DR+ CD4 T cells were similar in both age groups. In memory PB CD4 T cells, older age was only associated with increased CD57 expression. Significant age effects for LP CD8 T cells were only observed for CTLA-4 expression, with lower levels of expression observed on cells from older adults. Conclusions Greater age was associated with reduced expression of the co-inhibitory receptors CTLA-4 and PD-1 on LP CD4 T cells. Colonic LP CD8 T cells from older persons also displayed reduced CTLA-4 expression. These age-associated profiles were not observed in older PB memory CD4 T cells. The decline in co-inhibitory receptor expression on colonic LP T cells may contribute to local and systemic inflammation via a reduced ability to limit ongoing T cell responses to enteric microbial challenge.

Establishment of Neospora caninum antigen-specific T cell lines of primarily CD4+ T cells

2004 ◽  
Vol 26 (5) ◽  
pp. 243-246 ◽  
Author(s):  
W. Tuo ◽  
W. C. Davis ◽  
R. Fetterer ◽  
M. Jenkins ◽  
P. C. Boyd ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd4 T Cells ◽  
Neospora Caninum ◽  
T Cell Lines

scholarly journals 739 Intratumor childhood vaccine-specific CD4+ T cell recall helps antitumor CD8 T cells

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A770-A770
Author(s):  
Michael Brown ◽  
Zachary McKay ◽  
Yuanfan Yang ◽  
Darell Bigner ◽  
Smita Nair ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Viral Replication ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Antitumor Efficacy ◽  
Polio Vaccine ◽  
Recall Antigen ◽  
Childhood Vaccine

BackgroundPVSRIPO, a recombinant poliovirus derived from the live-attenuated Sabin oral polio vaccine strain, is being tested in multi-institutional phase II clinical trials for recurrent glioblastoma (NCT04479241) and unresectable, PD-1 refractory melanoma (NCT04577807) in combination with PD1 blockade. PVSRIPO capsid is identical to the Sabin vaccine strain and >99% identical to the inactivated Polio vaccine (IPOL, Salk), against which public health mandated childhood vaccination is near universal. In non-vaccinated mice, PVSRIPO mediates antitumor efficacy in a replication-dependent manner via engaging innate inflammation and antitumor T cells. Accordingly, it is anticipated that pre-existing immunity to PVSRIPO impedes antitumor therapy. However, recent evidence indicates that immunological 'recall', or reactivation of memory T cells, may mediate anti-tumor effects.MethodsThe impact of prior polio vs control (KLH) vaccination on intratumor viral replication, tumor inflammation, and overall tumor growth after intratumor PVSRIPO therapy was assessed in murine tumor models. The role of polio capsid and tetanus recall antigens in mediating intratumor inflammation and antitumor efficacy was similarly studied in mice non-permissive to PVSRIPO infection. To mechanistically define antitumor effects of polio recall, B cell and CD8 T cell knockout mice were used, in addition to adoptive transfer of CD4+ T cells from vaccinated mice. Intratumor polio or tetanus recall antigen therapy was performed after OT-I transfer (OVA-specific T cells) in the B16-OVA melanoma model to gauge antitumor T cell activity. Lastly, the inflammatory effects of polio and tetanus antigens was tested in human peripheral blood mononuclear cells (PBMCs).ResultsDespite curtailing intratumor viral replication, prior polio vaccination in mice potentiated subsequent antitumor efficacy of PVSRIPO. Intratumor recall responses induced by polio and tetanus antigens also delayed tumor growth. Recall antigen therapy was associated with marked intratumor influx of eosinophils, conventional CD4+ T cells, and increased expression of IFN-g, TNF, and Granzyme B in tumor infiltrating T cells. The antitumor efficacy of polio recall antigen was mediated by CD4+ T cells, partially depended upon CD8+ T cells, and was impaired by B cells. Both polio and tetanus recall antigen therapy bolstered the antitumor function of tumor-specific OT-I CD8+ T cells. Polio and tetanus antigens induced CXCL10 and type I/II/III IFNs in PBMCs in vitro.ConclusionsChildhood vaccine-specific CD4+ T cells hold cancer immunotherapy potential. In the context of PVSRIPO therapy, antitumor and inflammatory effects of polio vaccine-specific CD4+ T cell recall supersedes inhibitory effects of attenuated intratumor viral replication, and represents a novel mechanism of action.Ethics ApprovalThe animal work described in this study was approved by the Duke University IACUC.

scholarly journals CD4 T cell-intrinsic role for the T helper 17 signature cytokine IL-17: Effector resistance to immune suppression

2020 ◽  
Vol 117 (32) ◽  
pp. 19408-19414 ◽  
Author(s):  
Michael P. Crawford ◽  
Sushmita Sinha ◽  
Pranav S. Renavikar ◽  
Nicholas Borcherding ◽  
Nitin J. Karandikar
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Immune Suppression ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
T Helper ◽  
T Helper 17 ◽  
Immune Resistance ◽  
Inflammatory Bowel

Untoward effector CD4+ T cell responses are kept in check by immune regulatory mechanisms mediated by CD4+ and CD8+ T cells. CD4+ T helper 17 (Th17) cells, characterized by IL-17 production, play important roles in the pathogenesis of autoimmune diseases (such as arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, among others) and in the host response to infection and cancer. Here, we demonstrate that human CD4+ T cells cells exposed to a Th17-differentiating milieu are significantly more resistant to immune suppression by CD8+ T cells compared to control Th0 cells. This resistance is mediated, in part, through the action of IL-17A, IL-17F, and IL-17AF heterodimer through their receptors (IL-17RA and IL-17RC) on CD4+ T cells themselves, but not through their action on CD8+ T cells or APC. We further show that IL-17 can directly act on non-Th17 effector CD4+ T cells to induce suppressive resistance, and this resistance can be reversed by blockade of IL-1β, IL-6, or STAT3. These studies reveal a role for IL-17 cytokines in mediating CD4-intrinsic immune resistance. The pathways induced in this process may serve as a critical target for future investigation and immunotherapeutic intervention.

scholarly journals IL-21 from high-affinity CD4 T cells drives differentiation of brain-resident CD8 T cells during persistent viral infection

2020 ◽  
Vol 5 (51) ◽  
pp. eabb5590 ◽  
Author(s):  
Heather M. Ren ◽  
Elizabeth M. Kolawole ◽  
Mingqiang Ren ◽  
Ge Jin ◽  
Colleen S. Netherby-Winslow ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Viral Infection ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Central Nervous System Infection ◽  
Cell Receptor ◽  
Cd4 T Cell ◽  
Persistent Viral Infection ◽  
High Affinity

Development of tissue-resident memory (TRM) CD8 T cells depends on CD4 T cells. In polyomavirus central nervous system infection, brain CXCR5hi PD-1hi CD4 T cells produce interleukin-21 (IL-21), and CD8 T cells lacking IL-21 receptors (IL21R−/−) fail to become bTRM. IL-21+ CD4 T cells exhibit elevated T cell receptor (TCR) affinity and higher TCR density. IL21R−/− brain CD8 T cells do not express CD103, depend on vascular CD8 T cells for maintenance, are antigen recall defective, and lack TRM core signature genes. CD4 T cell–deficient and IL21R−/− brain CD8 T cells show similar deficiencies in expression of genes for oxidative metabolism, and intrathecal delivery of IL-21 to CD4 T cell–depleted mice restores expression of electron transport genes in CD8 T cells to wild-type levels. Thus, high-affinity CXCR5hi PD-1hi CD4 T cells in the brain produce IL-21, which drives CD8 bTRM differentiation in response to a persistent viral infection.

Generation of Cytomegalovirus-Specific T Lymphocytes Using Protein-Spanning Pools of pp65-Derived Over-Lapping Pentadecapeptides for Adoptive Immunotherapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2236-2236
Author(s):  
Guenther Koehne ◽  
Deepa Trivedi ◽  
Roxanne Y. Williams ◽  
Richard J. O’Reilly
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Lines ◽  
Cd8 T Cells ◽  
Adoptive Immunotherapy ◽  
Hla Class I ◽  
Class Ii ◽  
Hla Alleles ◽  
Hla Genotypes ◽  
T Cell Lines

Abstract Cell-mediated immunity is essential for control of human cytomegalovirus (HCMV) infection. We utilized a pool of 138 synthetic overlapping pentadecapeptides over-spanning the entire pp65 protein to generate polyclonal CMV-specific T-cell lines from 12 CMV-seropositive donors inheriting different HLA genotypes. Autologous monocyte-derived dendritic cells (DCs) pulsed with this complete pool consistently induced highly specific T-cells and in analyses of T-cell lines from 5 separate HLA-A*0201+ individuals demonstrate that this pp65-derived pentadecapeptide-pool selectively induced T-cells specifically reactive against sub-pools of pentadecapeptides which contained the HLA-A*0201 binding epitope NLVPMVATV. The specificity of these T-cells for this immunodominant nonapeptide was confirmed by MHC-tetramer staining and intracellular interferon-γ production, demonstrating that 38 – 60% of the CD8+ cell population were specific for this A*2-restricted peptide after 3 weeks of culture. These T cells also killed both nonapeptide-pulsed and CMV-infected target cells. In subsequent experiments using auotlogous monocyte-derived DC’s pulsed with the pentadecapeptide pool for the stimulation of CMV-specific T-cell lines in individuals other than HLA-A*2, the generated T cells selectively recognized 1–3 pentadecapeptides identified by secondary responses to a mapping grid of pentadecapeptide subpools with single overlaps. Responses against peptide loaded targets sharing single HLA class I or II alleles permitted the identification the restricting HLA alleles. Those T-cell lines from HLA-A*2 neg. donors contained high frequencies of CD4 and/or CD8 T-cells selectively reactive against peptides presented by other HLA alleles including known epitopes such as aa 341–350QYDPVAALF (HLA-A*2402) as well as unreported epitopes such as aa 267–275HERNGFTVL (HLA-B*4001 and B* 4002). In some donors, the peptide-specific IFN-g+ T-cells generated have been predominantly CD4+ T-cells. Like the peptide-specific CD8+ T-cells, we could determine both epitope and HLA-class II restricting element, e.g. aa513–523 FFWDANDIYRI (HLA-DRB1* 1301). These CD4+ T-cells also consistently exhibited cytotoxic activity against infected targets as well as peptide-loaded cells expressing the restricting HLA class II allele. Thus, synthetic overlapping pentadecapeptides spanning the sequence of the immunodominant protein of CMV-pp65, when loaded on DCs can consistently stimulate the in vitro generation of CD8+ and CD4+ T-cell lines from seropositive donors of diverse HLA genotypes. These cell lines are selectively enriched for T-cells specific for a limited number of immunodominant epitopes each presented by a single HLA class I or class II allele. This approach fosters expansion and selection of HLA-restricted CMV-pp65-reactive T-cell lines of high specificity which also lyse CMV-infected targets and may have advantages for generating virus-specific T-cells for adoptive immunotherapy.

Augmentation of Therapeutic Tumor Vaccine Efficacy by Genetic Engineering of CD4+ T Cell Help for Tumor Vaccine-Reactive CD8+ T Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3175-3175
Author(s):  
Sanju Jalla ◽  
Erin McCadden ◽  
Jie Wang ◽  
Ephraim J. Fuchs ◽  
Katharine A. Whartenby
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Autologous Tumor Cell ◽  
Autologous Tumor ◽  
Tumor Bearing ◽  
Cd4 T Cell Help ◽  
T Cell Help

Abstract Since CD4+ T cell help has been proposed to be required for maintaining the activity of tumor-specific CD8+ T cells, tolerance in tumor-specific CD4+ T cells may seriously impair the efficacy of therapeutic tumor vaccines. To overcome this problem, we devised a strategy to “engineer” CD4+ T cell help by treating tumor-bearing animals with nonmyeloablative conditioning and transplantation of autologous hematopoietic stem cells (HSCs) that have been genetically modified, via lentiviral transduction, to express an antigen containing “foreign” CD4+ T cell epitopes. After hematopoietic reconstitution, animals received the combination of an autologous tumor cell vaccine and an infusion of primed CD4+ T cells specific for the expressed epitopes. Using influenza hemagglutinin (HA) as the model antigen, we first confirmed that transplantation of HA-transduced HSCs led to efficient expression of HA by antigen-presenting cells, as demonstrated by the clonal expansion of adoptively transferred, HA-specific CD4+ transgenic T cells in mice receiving HA-transduced HSCs but not in mice receiving nerve growth factor receptor (NGFR) gene-transduced HSCs. Next, BALB/c mice harboring 13 day old, metastatic 4T1 mammary cancer were treated with removal of the primary, nonmyeloablative conditioning and transplantation of HA-transduced syngeneic HSCs, and following hematopoietic reconstitution, with concomitant autologous tumor cell vaccination and adoptive transfer of in vitro activated, HA-specific transgenic CD4+ T cells. This therapy was successful in curing the majority of tumor bearing mice, and was superior to the same therapy given to mice transplanted with NGFR-transduced stem cells. Finally, we found that the anti-tumor effect of vaccination plus exogenous T cell help was abolished by the adoptive transfer of either CD4+ or CD8+ T cells from tumor-bearing mice, suggesting that tumor-bearing mice contain both potential effectors and suppressors of anti-tumor immunity, the latter of which are abolished by the non-myeloablative conditioning. These results highlight the importance of CD4+ T cell help in the induction of therapeutic anti-tumor immunity.

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