TRAF2 regulates T cell immunity by maintaining a Tpl2-ERK survival signaling axis in effector and memory CD8 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.

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DC-Based Vaccines for Cancer Immunotherapy

Vaccines ◽

10.3390/vaccines8040706 ◽

2020 ◽

Vol 8 (4) ◽

pp. 706

Author(s):

Chunmei Fu ◽

Li Zhou ◽

Qing-Sheng Mi ◽

Aimin Jiang

Keyword(s):

Clinical Trials ◽

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Cd8 T Cells ◽

Critical Role ◽

Cd8 T Cell ◽

T Cell Immunity ◽

Cell Responses ◽

Cell Immunity

As the sentinels of the immune system, dendritic cells (DCs) play a critical role in initiating and regulating antigen-specific immune responses. Cross-priming, a process that DCs activate CD8 T cells by cross-presenting exogenous antigens onto their MHCI (Major Histocompatibility Complex class I), plays a critical role in mediating CD8 T cell immunity as well as tolerance. Current DC vaccines have remained largely unsuccessful despite their ability to potentiate both effector and memory CD8 T cell responses. There are two major hurdles for the success of DC-based vaccines: tumor-mediated immunosuppression and the functional limitation of the commonly used monocyte-derived dendritic cells (MoDCs). Due to their resistance to tumor-mediated suppression as inert vesicles, DC-derived exosomes (DCexos) have garnered much interest as cell-free therapeutic agents. However, current DCexo clinical trials have shown limited clinical benefits and failed to generate antigen-specific T cell responses. Another exciting development is the use of naturally circulating DCs instead of in vitro cultured DCs, as clinical trials with both human blood cDC2s (type 2 conventional DCs) and plasmacytoid DCs (pDCs) have shown promising results. pDC vaccines were particularly encouraging, especially in light of promising data from a recent clinical trial using a human pDC cell line, despite pDCs being considered tolerogenic and playing a suppressive role in tumors. However, how pDCs generate anti-tumor CD8 T cell immunity remains poorly understood, thus hindering their clinical advance. Using a pDC-targeted vaccine model, we have recently reported that while pDC-targeted vaccines led to strong cross-priming and durable CD8 T cell immunity, cross-presenting pDCs required cDCs to achieve cross-priming in vivo by transferring antigens to cDCs. Antigen transfer from pDCs to bystander cDCs was mediated by pDC-derived exosomes (pDCexos), which similarly required cDCs for cross-priming of antigen-specific CD8 T cells. pDCexos thus represent a new addition in our arsenal of DC-based cancer vaccines that would potentially combine the advantage of pDCs and DCexos.

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Subunit Vaccine-Elicited Effector-Like Memory CD8 T Cells Protect Against Listeriosis

10.1101/2020.12.01.407577 ◽

2020 ◽

Author(s):

Woojong Lee ◽

Autumn Larsen ◽

Brock Kingstad-Bakke ◽

M. Suresh

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Protective Immunity ◽

Cd8 T Cell ◽

T Cell Memory ◽

Memory Cd8 T Cells ◽

Cell Immunity ◽

Nano Emulsion ◽

Cd8 T Cell Memory

AbstractDevelopment of T-cell-based subunit protein vaccines against diseases, such as AIDS, tuberculosis and malaria remains a challenge for immunologists. Here, we have evaluated whether cross-presentation induced by nanoemulsion adjuvant Adjuplex (ADJ), can be combined with the immunomodulatory effects of TLR agonists (CpG or glucopyranosyl lipid adjuvant [GLA]) to evoke protective systemic CD8 T cell-based immunity to Listeria monocytogenes (LM). Vaccination with ADJ, alone or in combination with CpG or GLA augmented activation and antigen uptake by migratory and resident dendritic cells and up-regulated CD69 expression on B and T lymphocytes in draining lymph nodes. By virtue of its ability to engage BATF3-dependent cross-presenting DCs, ADJ potently elicited effector CD8 T cells that differentiated into a distinct subset of granzyme B-expressing CD27LO effector-like memory CD8 T cells, which provided highly effective immunity to LM in spleen and liver. CpG or GLA alone did not elicit effector-like memory CD8 T cells and induced moderate protection in spleen, but not in the liver. Surprisingly, combining CpG or GLA with ADJ limited the magnitude of ADJ-induced CD8 T cell memory and compromised protective immunity to LM, especially in the liver. Taken together, data presented in this manuscript provides a glimpse of protective CD8 T cell memory differentiation induced by a nano-emulsion adjuvant and demonstrates the unexpected negative effects of TLR signaling on the magnitude of CD8 T cell memory and protective immunity to listeriosis.ImportanceTo date, the most effective vaccines primarily provide protection by eliciting neutralizing antibodies, while development of T-cell-based subunit vaccines against infectious diseases, such as tuberculosis and malaria, remains a challenge for immunologists. Axiomatically, engagement of multiple innate immune receptors early in the response might be key to programming effective immunity. Hence, there is an impetus to develop combination adjuvants that engage multiple innate signaling pathways and additionally promote cross-presentation to stimulate CD8 T-cell immunity. Here, we show that a nano-emulsion adjuvant ADJ alone elicits effector-like memory CD8 T cells and provides highly effective immunity to listeriosis; combining ADJ with TLR agonists, including CpG and GLA, compromised T cell immunity to LM. In summary, this study provided fundamental insights into the effects of combining innate immune signaling with nano-emulsion adjuvants on memory T cell differentiation and protective immunity. These findings are expected to have implications in the use of combination adjuvants to develop subunit vaccines that engender systemic CD8 T-cell immunity to intracellular pathogens.

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Blocking VIP Signaling Abrogates Upregulation Of PD1 and Increases Proliferation Of Allo-Reactive T-Cells In a Mixed Lymphocyte Reaction

Blood ◽

10.1182/blood.v122.21.1041.1041 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1041-1041

Author(s):

Emily R Summerbell ◽

Cynthia R. Giver ◽

Sravanti Rangaraju ◽

Katarzyna Anna Darlak ◽

Edmund K. Waller

Keyword(s):

Flow Cytometry ◽

Cell Proliferation ◽

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

T Cell Immunity ◽

T Cell Proliferation ◽

Cell Immunity

Abstract Introduction Vasoactive intestinal peptide (VIP) is a neuropeptide hormone that suppresses Th1 immunity and inhibits antiviral immunity. Decreased Th1 immunity is problematic for allogeneic bone marrow transplant (allo-BMT) patients requiring T-cell immunity against blood cancers (Graft-versus-Tumor) and against secondary infections such as CMV. VIPhyb, a modified VIP peptide, is a VIP receptor antagonist that decreases VIP signaling. VIP-knockout mice and mice treated with VIPhyb after allo-BMT are known to have better antiviral immunity and survival after CMV infection without increasing GvHD (Li et al. PLoS One. 2013 May 27;8(5):e63381) (Li et al. Blood. 2013 Mar 21;121(12):2347-51.), thus making VIPhyb of interest for pharmacological use in humans to improve the efficacy of allo-BMT The effects of VIPhyb on T-cell immunity are not yet fully profiled. This study aimed to analyze the effects of VIPhyb on CD4+ and CD8+ T-cell proliferation and activation in order to better understand the mechanistic implications of VIP inhibition on T-cell adaptive immunity. This study also aimed to show that mixed lymphocyte reactions (MLRs), an in vitro allo-BMT model, could be used to provide rapid and reliable results that are consistent with in vivo data. It was hypothesized that VIPhyb would increase T-cell immunity as profiled by: increased T-cell proliferation, CD69 and PD1 co-upregulation in early T-cell activation, and PD1 downregulation in T-cells after initial activation. Methods Splenocytes from two histoincompatible mice were cultured together at 37°C in a 1:1 ratio in a one-way MLR. BALB/c splenocytes (stimulators) were irradiated at 20Gy, and Pepboy splenocytes (responders) were labeled with CFSE to trace proliferation. VIPhyb was added daily to the cell cultures in doses of 0.1μM, 0.3μM, 1μM, or 3μM. Treatment groups were compared to a PBS control. Proliferation, CD69, and PD1 were assessed by flow cytometry on the BD FACSAria. All results are shown as mean ± SEM (n=3). One-way ANOVA tests with Dunnett post-tests were calculated using Prism software. *p < 0.05; **p < 0.01; ***p < 0.001 Results VIPhyb increased CD4+ and CD8+ T-cell proliferation: 3, 5, and 7 days after initiating a one-way MLR, CFSE expression of Pepboy responder T-cells was assessed using flow cytometry (Figure 1). As the VIPhyb dose increased, the percentage of initial splenocytes that underwent proliferation increased in both CD4+ and CD8+ T-cells. VIPhyb increased early T-cell CD69 expression and abrogated later PD1 upregulation in CD8+ T-cells: 3, 5, and 7 days after initiating a one-way MLR, expression levels of CD69 and PD1 on Pepboy responder T-cells were assessed by flow cytometry. Significant upregulation of CD69 on CD4+ and CD8+ T-cells on day 3 occurred with increasing VIPhyb doses (Figures 2A and 2B). PD1 was co-upregulated with CD69 during early activation, and VIPhyb significantly decreased PD1 expression on CD8+ T-cells on days 5 and 7 (Figures 2C and 2D). Conclusions VIPhyb increased T-cell proliferation; CD8+ T-cells were affected more significantly. VIPhyb increased early co-upregulation of CD69 and PD1 in all T-cells and significantly decreased later CD8+ T-cell PD1 expression, indicating that VIPhyb increases T-cell activation. We hypothesize that the decreased PD1 expression will be critical for understanding the pathways involved in VIP inhibition. Importantly, since it has been shown in vivo that VIPhyb does not increase GvHD, then it can be assumed that the VIPhyb-induced T-cell proliferation and activation will increase GvL and adaptive immunity without increasing alloreactivity. Notably, these results are consistent with published in vivo data, which demonstrates that the MLR can be used as a faster method of analyzing pharmacological compounds than in vivo experiments. Given these results, VIPhyb is still of interest as a potential therapy for allo-BMT patients. Disclosures: No relevant conflicts of interest to declare.

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Cross-priming CD8+ T cells by targeting antigens to human dendritic cells through DCIR

Blood ◽

10.1182/blood-2010-01-264960 ◽

2010 ◽

Vol 116 (10) ◽

pp. 1685-1697 ◽

Cited By ~ 138

Author(s):

Eynav Klechevsky ◽

Anne-Laure Flamar ◽

Yanying Cao ◽

Jean-Philippe Blanck ◽

Maochang Liu ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

T Cell ◽

Cd8 T Cells ◽

T Cell Responses ◽

Cd8 T Cell ◽

T Cell Immunity ◽

Cross Presentation ◽

Cell Responses ◽

Cell Immunity

Abstract We evaluated human CD8+ T-cell responses generated by targeting antigens to dendritic cells (DCs) through various lectin receptors. We found the immunoreceptor tyrosine-based inhibitory motif-containing DC immunoreceptor (DCIR) to mediate potent cross-presentation. A single exposure to a low dose of anti-DCIR–antigen conjugate initiated antigen-specific CD8+ T-cell immunity by all human DC subsets including ex vivo–generated DCs, skin-isolated Langerhans cells, and blood myeloid DCs and plasmacytoid DCs. The delivery of influenza matrix protein (FluMP) through DCIR resulted in expansion of FluMP-specific memory CD8+ T cells. Enhanced specific CD8+ T-cell responses were observed when an antigen was delivered to the DCs via DCIR, compared with those induced by a free antigen, or antigen conjugated to a control monoclonal antibody or delivered via DC-SIGN, another lectin receptor. DCIR targeting also induced primary CD8+ T-cell responses against self (MART-1) and viral (HIV gag) antigens. Addition of Toll-like receptor (TLR) 7/8 agonist enhanced DCIR-mediated cross-presentation as well as cross-priming, particularly when combined with a CD40 signal. TLR7/8 activation was associated with increased expansion of the primed CD8+ T cells, high production of interferon-γ and tumor necrosis factor-α, and reduced levels of type 2–associated cytokines. Thus, antigen targeting via the human DCIR receptor allows activation of specific CD8+ T-cell immunity.

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Specific CD8+ TCR Repertoire Recognizing Conserved Antigens of SARS-CoV-2 in Unexposed Population: A Prerequisite for Broad-Spectrum CD8+ T Cell Immunity

Vaccines ◽

10.3390/vaccines9101093 ◽

2021 ◽

Vol 9 (10) ◽

pp. 1093

Author(s):

Wei Hu ◽

Meifang He ◽

Xiaoning Wang ◽

Qiang Sun ◽

Ming Kuang

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

Broad Spectrum ◽

Cd8 T Cell ◽

T Cell Immunity ◽

Healthy Population ◽

Cell Immunity ◽

Wide Range ◽

Tcr Repertoire

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has developed variants escaping neutralization antibody immunity established against the original virus. An understanding of broad-spectrum adaptive immunity, including CD8+ T cell immunity to wide range of epitopes, could help translational efforts to improve coronavirus disease 2019 (COVID-19) prevention and therapy. However, there have been few direct studies in which such immunity exists in a population. Methods: We selected SARS-CoV-2-conserved structural peptides that are not prone to mutation as antigens for broad-spectrum CD8+ T cell immunity. Peripheral blood mononuclear cells (PBMCs) from unexposed healthy donors were stimulated with these peptides in vitro and CD8+ T cell-specific response was monitored. The conserved peptide-specific CD8+ T cells were sorted for T cell receptor (TCR) repertoire sequencing. The presence of specific complementary determining region 3 (CDR3) clones was analyzed in a healthy cohort. Results: For each structural protein, including S, E, M, N, the conserved peptides could potentially provide the largest number of major histocompatibility complex-I (MHC-I) epitopes in the Oriental and Caucasian populations. For conserved peptides from spike (S), envelope (E), membrane (M), nucleocapsid (N) proteins, we found that there were no cross-reactive memory T cells in the unexposed individuals. Instead, their T cells contain naïve TCR repertoire recognizing these conserved peptides. Using TCR sequencing and CDR3 clustering for the conserved peptides specific T cells, we found that the recovered patients had a higher proportion of TCR repertoire similar with that of specific CD8+ T cells in unexposed individuals. Meanwhile, CDR3 clones of the above T cells were widely present in the healthy population. Conclusions: This study provides evidence of broad-spectrum SARS-CoV-2 specific CD8+ TCR repertoire in unexposed healthy population, which is implicated in the development and implementation of broad-spectrum vaccines against COVID-19.

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Differential Longevity of Memory CD4 and CD8 T Cells in a Cohort of the Mothers With a History of ZIKV Infection and Their Children

Frontiers in Immunology ◽

10.3389/fimmu.2021.610456 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jessica Badolato-Corrêa ◽

Fabiana Rabe Carvalho ◽

Iury Amancio Paiva ◽

Débora Familiar-Macedo ◽

Helver Gonçalves Dias ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

T Cell Responses ◽

T Cell Immunity ◽

Zikv Infection ◽

Cell Responses ◽

Cell Immunity ◽

History Of ◽

Ifn Γ

Background: Zika virus (ZIKV) infection causes for mild and self-limiting disease in healthy adults. In newborns, it can occasionally lead to a spectrum of malformations, the congenital Zika syndrome (CZS). Thus, little is known if mothers and babies with a history of ZIKV infection were able to develop long-lasting T-cell immunity. To these issues, we measure the prevalence of ZIKV T-cell immunity in a cohort of mothers infected to the ZIKV during pregnancy in the 2016–2017 Zika outbreak, who gave birth to infants affected by neurological complications or asymptomatic ones.Results: Twenty-one mothers and 18 children were tested for IFN-γ ELISpot and T-cell responses for flow cytometry assays in response to CD4 ZIKV and CD8 ZIKV megapools (CD4 ZIKV MP and CD8 ZIKV MP). IFN-γ ELISpot responses to ZIKV MPs showed an increased CD4 and CD8 T-cell responses in mothers compared to children. The degranulation activity and IFN-γ-producing CD4 T cells were detected in most mothers, and children, while in CD8 T-cells, low responses were detected in these study groups. The total Temra T cell subset is enriched for IFN-γ+ CD4 T cells after stimulation of CD4 ZIKV MP.Conclusion: Donors with a history of ZIKV infection demonstrated long-term CD4 T cell immunity to ZIKV CD4 MP. However, the same was not observed in CD8 T cells with the ZIKV CD8 MP. One possibility is that the cytotoxic and pro-inflammatory activities of CD8 T cells are markedly demonstrated in the early stages of infection, but less detected in the disease resolution phase, when the virus has already been eliminated. The responses of mothers' T cells to ZIKV MPs do not appear to be related to their children's clinical outcome. There was also no marked difference in the T cell responses to ZIKV MP between children affected or not with CZS. These data still need to be investigated, including the evaluation of the response of CD8 T cells to other ZIKV peptides.

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Negative effect of CTLA-4 on induction of T-cell immunity in vivo to B7-1+, but not B7-2+, murine myelogenous leukemia

Blood ◽

10.1182/blood.v99.6.2146 ◽

2002 ◽

Vol 99 (6) ◽

pp. 2146-2153 ◽

Cited By ~ 34

Author(s):

James L. LaBelle ◽

Carrie A. Hanke ◽

Bruce R. Blazar ◽

Robert L. Truitt

Keyword(s):

T Cells ◽

T Cell ◽

Cd8 T Cells ◽

T Cell Immunity ◽

Myelogenous Leukemia ◽

Cell Immunity ◽

B7 Molecules ◽

Negative Effect

Abstract B7 molecules provide important costimulatory signals to T cells, and B7 genes have been introduced into B7-negative tumor cells to enhance their immunogenicity. However, the role of B7 molecules in inducing tumor immunity is controversial because of conflicting results and reports of differential signaling through the B7 molecules and their ligands CD28 and CTLA-4. In this study, we compared the effect of B7-1 (CD80) and B7-2 (CD86) on the induction of T-cell immunity to C1498, a murine myelogenous leukemia. When cultured with exogenous cytokines in vitro, C1498/B7-1 and C1498/B7-2 induced syngeneic CD8+ T cells to kill parental C1498. In vivo, C1498/B7-1 grew progressively after subcutaneous injection, whereas C1498/B7-2 completely regressed after transient growth in naive mice. Spontaneous rejection of C1498/B7-2 resulted in immunity to challenge doses of C1498 and C1498/B7-1. Antibody-depletion studies in vivo showed that CD8+ T cells rejected C1498/B7-2, whereas only natural killer cells affected the growth of C1498/B7-1. Two approaches were used to determine whether preferential interaction of B7-1 with CTLA-4 contributed to the failure of C1498/B7-1 to activate CD8+ T cells in vivo. First, CTLA-4 specific monoclonal antibody was used to block B7-1–CTLA-4 interaction. Second, CTLA-4 deletional mutant (−/−) bone marrow chimeras were used as tumor hosts. In both systems, there was a significant increase in the rate of rejection of C1498/B7-1 tumors. Resistance to C1498/B7-1 in CTLA-4−/− hosts was mediated by CD8+ T cells. Blocking or deletion of CTLA-4 did not affect the growth of parental C1498, indicating that B7-1 was important for the induction of CD8+ T-cell immunity in the absence of CTLA-4.

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IL-17RA-signaling modulates CD8+ T cell survival and exhaustion during Trypanosoma cruzi infection

10.1101/314336 ◽

2018 ◽

Cited By ~ 1

Author(s):

Jimena Tosello Boari ◽

Cintia L. Araujo Furlan ◽

Facundo Fiocca Vernengo ◽

Constanza Rodriguez ◽

María C. Ramello ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Trypanosoma Cruzi ◽

Cell Survival ◽

Cd8 T Cells ◽

Cd8 T Cell ◽

T Cell Immunity ◽

Cell Immunity ◽

T Cell Survival

AbstractThe IL-17 family contributes to host defense against many intracellular pathogens by mechanisms not fully understood. CD8+ T lymphocytes are key elements against intracellular microbes and their survival and appropriate response is orchestrated by several cytokines. Here, we demonstrated that IL-17RA-signaling cytokines sustain pathogen-specific CD8+ T cell immunity. Absence of IL-17RA and IL-17A/F during Trypanosoma cruzi infection resulted in increased tissue parasitism and reduced frequency of parasite-specific CD8+ T cells. Impaired IL-17RA-signaling in vivo increased apoptosis of parasite-specific CD8+ T cells while recombinant IL-17 in vitro down-regulated the pro-apoptotic protein BAD and promoted activated CD8+ T cell survival. Phenotypic, functional and trancriptomic profiling showed that T. cruzi-specific CD8+ T cells arising in IL-17RA-deficient mice presented features of cell dysfunction. PD-L1 blockade partially restored the magnitude of CD8+ T cell responses and parasite control in these mice. Adoptive transfer experiments established that IL-17RA-signaling is intrinsically required for the proper maintenance of functional effector CD8+ T cells. Altogether, our results identify IL-17RA and IL-17A as critical factors for sustaining CD8+ T cell immunity to T. cruzi.

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NY-ESO-1 Specific T-Cells Are Spontaneously Present in High-Risk Myeloma and Kill Primary Myeloma Cells.

Blood ◽

10.1182/blood.v104.11.2454.2454 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2454-2454

Author(s):

Frits van Rhee ◽

Susann M. Szmania ◽

Amberly Moreno ◽

Ramesh Batchu ◽

Mindy Pomtree ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

High Risk ◽

Cd8 T Cells ◽

De Novo ◽

Myeloma Cell ◽

Cysteine Residue ◽

T Cell Immunity ◽

Myeloma Cells ◽

Cell Immunity

Abstract We have previously reported that the Cancer/Testis antigen NY-ESO-1 is frequently expressed in patients with high-risk myeloma or advanced myeloma characterized by cytogenetic abnormalities. We tested 3 HLA-A*0201 positive MM patients with abnormal cytogenetics for the spontaneous presence of NY-ESO-1 specific CD8+ T-cells in the peripheral blood. We were able to detect 0.1% and 0.6% NY-ESO-1 specific CD8+ T-cells respectively in 2 patients using HLA-A*0201/NY-ESO-1157-165 specific tetramers prior to any re-stimulation with peptide. The 3rd patient did not have NY-ESO-1 specific T-cells. This last patient had been very heavily pre-treated and in contrast to the other patients, influenza matrix peptide58–66 specific T-cells failed to expand indicating that this patient’s immune system may have been exhausted. CD3+/8+ T-cells were expanded in multiple experiments by re-stimulation with autologous monocyte derived dendritic cells (Mo-DCs) pulsed with NY-ESO-1157-C165V analogue peptide. Substitution of cysteine (C) for valine (V) at position 165 improves binding affinity for HLA-A*0201 and antigenicity by eliminating dimerization of the carboxy-terminus cysteine residue. The lines contained 45–79% tetramer positive T-cells after stimulation. The CTLs were highly specific and lysed NY-ESO-1 positive/ HLA-A*0201 positive primary myeloma cells (50%), the NY-ESO-1 positive/ HLA-*0201 positive myeloma cell line U266 (55%) and autologous PHA-blasts pulsed with NY-ESO-1157–165 wild-type peptide (75%), but not K562 or patient PHA-blasts pulsed with the HLA-A*0201 binding MAGE-3112–120 peptide at E:T ratios of 10:1. The CTLs were of Tc1 type (g-IFN positive and IL4 negative) and stained positive for granzymes suggesting killing via the perforin/granzyme pathway. Immunophenotyping showed that the CTLs expressed the IL2 receptor and the integrin VLA-4, which is associated with the ability to transgress endothelial barriers. Further, the CTLs predominantly belonged to the terminal effector compartment (CD45RO+/CCR7−). We were unable to detect or expand NY-ESO-1 specific T-cells in HLA-A*0201 positive normal donors or NY-ESO-1 negative MM patients suggesting that pre-existent T-cell immunity to NY-ESO-1 is a prerequisite for the expansion of NY-ESO-1 specific CTLs. It also ruled out the possibility that the use of autologous APC’s induced de novo NY-ESO-1 specific CTL. In conclusion, these data suggest that NY-ESO-1 positive myeloma tumors are able to elicit spontaneous T-cell immunity and that expanded CTLs from these patients can kill primary myeloma cells. These CTLs recognize on the cell surface of myeloma cells naturally processed and presented NY-ESO-1, which is displayed in the context of HLA-A*0201. These results suggest that patients with high-risk NY-ESO-1 positive myeloma have a pool of memory CTLs, which can be exploited for vaccine or other immuno-therapeutic approaches.

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