A Short CD3/CD28 Costimulation Combined with IL-21 Enhance the Generation of Human Memory Stem T Cells for Adoptive Immunotherapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5424-5424 ◽  
Author(s):  
Carmen Alvarez-Fernandez ◽  
Laura Escribà-Garcia ◽  
Jorge Sierra ◽  
Javier Briones
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Ex Vivo ◽  
Culture Period ◽  
Cell Subpopulation ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Cd28 Costimulation

Abstract Introduction: Adoptive transfer of gene modified T cells (ACT) with chimeric antigen receptors (CARs) is becoming a clinically relevant immunotherapy approach for cancer treatment. One important question involves the selection of the T cell subpopulation for gene-modified ACT. Recent preclinical studies have shown that memory stem T cells (TSCM) (with a phenotype CD45RO-, CD45RA+, CCR7+, CD62L+, CD95+) have higher in vivo persistence, self-renewal, proliferative and antitumor capacities compared to other memory and effector T cell subpopulations. For these reasons, TSCM may represent an ideal candidate for ACT with CARs. Ex vivo generation of TSCM rely on CD3/CD28 costimulation and the use of cytokines such as IL-7 and IL-15 during the entire culture period. However, a strong costimulation may induce differentiation of TSCM to effector memory T cells. In this study, we have analyzed the impact of the costimulation length and the addition of the IL-21 on the ex vivo generation of TSCM cells. Methods: Purified naïve T cells from healthy donors were cultured in the presence of anti-CD3/CD28 coated beads, IL-7, IL-15 and/or IL-21 (25ng/ml). T cells phenotype from the different memory (TCM, TSCM and TEM) and effector subpopulations were analyzed by multiparametric flow cytometry. Particularly, TSCM were identified by a specific plotting strategy. Briefly, CD4+ and CD8+ cells were gated and a CCR7 against CD45RO plot were used to select CCR7+ CD45RO- cells in each subpopulation. Within these cells, a CCR7+ CD45RO- CD45RA+ cell population was identified by plotting CCR7 against CD45RA. Then, CD27 was used to detect CCR7+ CD45RO- CD45RA+ CD27+ cells. Finally, a plot of CD95 against CCR7 was used to identify the CCR7+ CD45RO- CD45RA+ CD27+ CD95+ cell population. Cellular proliferation was analyzed by flow cytometry using CFSE. Results: A short (48 hours) anti-CD3/CD28 costimulation of naïve T cells combined with IL-7 and IL-15 significantly increased the frequencies of CD4+ and CD8+ TSCMex vivo, compared to a long (10 days) costimulation (34.6%±4.4% vs 15.6%±4.24% for CD4+; p=0.008, and 20.5%±4.00% vs 7.7%±2.53% for CD8+; p=0.02). Moreover, the addition of IL-21 to this condition further enhanced the enrichment (47.9%±4.1% vs 34.6%±4.4% for CD4+; p=0.006, and 34.1%±3.5% vs 20.5%±4% for CD8+; p<0.0005) and expansion of CD4+ and CD8+ TSCM, with an increase in absolute numbers (311.3±39 vs 192.8±58.6 fold expansion in CD4+; p=0.04, and 728.1±53 vs 442.7±122 fold expansion in CD8+; p=0.04). In addition, IL-21 did not preferentially enriched for the CD8+ population since the CD4+ TSCM were maintained over the cell culture period in all the conditions tested (CD4+/CD8+ TSCM ratio:1.03±0.28 vs 1.34±0.19 for long costimulation versus long + IL-21, and 0.68±0.14 vs 0.72±0.002 for short vs short + IL-21; p=0.23). Conclusion: We show that these refined in vitro conditions significantly increase the frequencies and expansion of TSCM. Since the reagents used are available in a clinical grade setting, these data may have relevant clinical implications for the generation of this memory T cell subset for adoptive cell therapy with CAR-T cells of patients with cancer. Disclosures No relevant conflicts of interest to declare.

scholarly journals Blinatumomab-Induced T Cell Activation at Single Cell Transcriptome Resolution

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3886-3886
Author(s):  
Hong Yin ◽  
Yi Huo ◽  
Zhen Sheng ◽  
Chi-Ming Li ◽  
Daniel C Ellwanger ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Single Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Ex Vivo ◽  
Target Cells ◽  
Ex Vivo Model ◽  
Naive T Cells ◽  
Naïve T Cells

Introduction Blinatumomab, a bi-specific T cell engager (BiTE®) combining the VH and VL domains of two antibodies against human CD19 and CD3, has been approved by U.S. Food and Drug Administration (FDA) for the treatment of patients with relapsed or refractory B precursor ALL (r/r B-ALL) for its clinical benefit demonstrated in different clinical trials. Clinical trials have also shown that there are still patients refractory to blinatumomab. It is thus important to understand the resistance mechanisms. Blinatumomab connects patients' autologous T cells and target cells to form immunologic synapse which potently triggers the activation signaling cascades in T cells and guides T cells to recognize and induce perforin/granzyme-mediated lysis of CD19+ B-ALL cells. Previous studies showed blinatumomab-mediated cytotoxicity involves different T cell subpopulations. But response of each T cell subpopulation to blinatumomab treatment remained largely unknown. Methods and Results In this study, we used 10X Genomics based single cell RNA sequencing to analyze the transcriptome of single T cells before and after blinatumomab treatment. First, ex vivo blinatumomab cytotoxicity model was established, in which healthy PBMCs were used as effector cells and cocultured with target cells (RS4;11 cells or SUP-B15 cells) at an effector-to-target cell ratio of 10:1 with 0.1 ng/mL blinatumomab. Then, PBMCs and BMMCs from 2 B-ALL patients were cultured with 10 ng/mL blinatumomab. Cells from both ex vivo model and patient samples were sequenced using 10X Genomics platform. In total, transcriptome of 17920 single T cells from the ex vivo model and 2271 single T cells from patient sample were analyzed. Based on T cell trajectory analysis, we identified four distinct populations of blinatumomab-activated T cells, which were derived from CD8+ effector memory T (TEM) cells, CD4+ central memory (TCM) cells, naïve T cells and Tregs, respectively. The differentially expressed genes in activated clusters were analyzed to reflect T cell activation mechanisms. The result showed blinatumomab induced the upregulation of aerobic glycolysis pathway (PKM, PGAM1, ENO1, GAPDH and LDHA), cytoskeleton dynamics pathway (ACTD1, ACTB, NME1 and TUBA1B), IFN-responsive pathway (GBP1, PSME2, WARS, CXCL10 and STAT1), and the upregulation of well-known immune-related genes (TNFRSF4, TNFRSF18, LAG3, CD69, IL2RA, MIR155HG, BATF, SH2D2A, LTA, NFKBIA and NDFIP2). We found blinatumomab-activated CD8+ TEM cells showed stronger cytotoxic capability than other activated populations with specific production of cytotoxic factors (PRF1, IFNG and FASLG) and cytokines (CCL2, CCL3, CCL3L1, CCL4, CCL4L2, CCL8, XCL1, XCL2, TNFSF9 and TNFSF14). Last, differential gene expression analysis revealed that co-stimulatory (TNFRSF4,TNFRSF9 and TNFRSF18) and co-inhibitory receptors (LAG3 and TIGIT) were similarly up-regulated in clusters activated from memory and naïve T cells, indicating ligand dependent T cell functional outcomes induced by blinatumomab. Conclusion In summary, we used single cell sequencing to map the blinatumomab-mediated T cell activation state transition and reveal the molecular changes in different T cell subpopulations. Memory T cells, naïve T cells and Tregs were identified functional populations after blinatumomab treatment. CD8+ TEM accounted for the majority of blinatumomab-induced cytotoxicity. Furthermore, T cell co-regulatory receptors were identified as potential targets accountable for blinatumomab sensitivity or resistance mechanisms. The study demonstrated that the collected cellular transcriptional profiles can serve as resource to explore novel strategies to enhance the efficacy of blinatumomab. Disclosures Yin: Amgen: Employment. Huo:Amgen: Employment. Sheng:Amgen: Employment. Li:Amgen: Employment. Ellwanger:Amgen: Employment. Lu:Amgen: Employment. Homann:Amgen: Employment. Wang:Amgen: Employment. Ren:Ruijin hospital: Employment.

Engineering Mesenchymal Cells with Interleukin 7 Gene: In Vitro Effects on Naive T Cell Population.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5135-5135
Author(s):  
Paolo Sportoletti ◽  
Beatrice Del Papa ◽  
Mariangela De Ioanni ◽  
Lorenzo Moretti ◽  
Elisabetta Bonifacio ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Population ◽  
Mesenchymal Cells ◽  
Independent Effect ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Interleukin 7 ◽  
Naïve T Cell ◽  
Naive T Cell

Abstract T cell homeostasis is regulated by several molecules among which Interleukin 7 (IL-7) plays an essential role for survival and homeostatic proliferation of peripheral naive T cells. In a previous study we demonstrated whether human mesenchymal cells could be engineered with IL-7 gene to produce functional level of this cytokine. Now we analysed the impact of different quantities of IL-7 produced by mesenchymal cells on survival and proliferation of a negative immunoselected naive (CD3+/CD45RA+) T cell population. Co-cultivation of peripheral naive T cells with mesenchymal cells producing low (16 pg/ml) or high (1000 pg/ml) IL-7 levels or in presence of exogenous IL-7 (0,01 ng/ml and 100 ng/ml) maintained the CD3+/CD45RA+ naive T cell phenotype. The chemokine receptor CCR7+ expression was also maintained among this T cell population. Naive T cell molecular characteristics were maintained as assessed by the Vβ spectratyping complexity score which shows the maintenance of a broad T cell repertoire. No Th1 or Th2 differentiation was observed as assessed by IFNγ or IL-4 accumulation. In contrast only mesenchymal cells producing high IL-7 amount caused increases in the activation (CD25 31.2%±12 vs 10%±3.5, p&lt;0.05), proliferation (CD71 17.8±7% vs 9.3%±3, p&lt;0.05), apoptosis (assessed by annexin V: 18.6%±5 vs 14.9%±2.6, p&gt;0.05) and phase S cell cycle (15% vs 6.9%, p&gt;0.05). Exogenous IL-7 did not exert any significant effect. In conclusion, we demonstrated that IL-7 produced by mesenchymal cells has a dose-independent effect on naive T cell survival while exerts a dose-dependent effect on activation/proliferation. Due to continuous production of IL-7 by engineered cells, our system emerge as more efficacious than the exogenous IL-7.

scholarly journals Antigen-dependent and -independent Ca2+ Responses Triggered in T Cells by Dendritic Cells Compared with B Cells

1998 ◽  
Vol 188 (8) ◽  
pp. 1473-1484 ◽  
Author(s):  
Jérôme Delon ◽  
Nadège Bercovici ◽  
Graça Raposo ◽  
Roland Liblau ◽  
Alain Trautmann
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
B Cells ◽  
Mhc Class Ii ◽  
Ex Vivo ◽  
Cell Formation ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Survival Signal

Dendritic cells (DCs) are much more potent antigen (Ag)-presenting cells than resting B cells for the activation of naive T cells. The mechanisms underlying this difference have been analyzed under conditions where ex vivo DCs or B cells presented known numbers of specific Ag–major histocompatibility complex (MHC) complexes to naive CD4+ T cells from T cell antigen receptor (TCR) transgenic mice. Several hundred Ag–MHC complexes presented by B cells were necessary to elicit the formation of a few T–B conjugates with small contact zones, and the resulting individual T cell Ca2+ responses were all-or-none. In contrast, Ag-specific T cell Ca2+ responses can be triggered by DCs bearing an average of 30 Ag–MHC complexes per cell. Formation of T–DC conjugates is Ag-independent, but in the presence of the Ag, the surface of the contact zone increases and so does the amplitude of the T cell Ca2+ responses. These results suggest that Ag is better recognized by T cells on DCs essentially because T–DC adhesion precedes Ag recognition, whereas T–B adhesion requires Ag recognition. Surprisingly, we also recorded small Ca2+ responses in T cells interacting with unpulsed DCs. Using DCs purified from MHC class II knockout mice, we provide evidence that this signal is mostly due to MHC–TCR interactions. Such an Ag-independent, MHC-triggered calcium response could be a survival signal that DCs but not B cells are able to deliver to naive T cells.

scholarly journals Inducible RNAi in vivo reveals that the transcription factor BATF is required to initiate but not maintain CD8+ T-cell effector differentiation

2014 ◽  
Vol 112 (2) ◽  
pp. 512-517 ◽  
Author(s):  
Jernej Godec ◽  
Glenn S. Cowley ◽  
R. Anthony Barnitz ◽  
Ozan Alkan ◽  
David E. Root ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cell Fate ◽  
Viral Vectors ◽  
Ex Vivo ◽  
Experimental System ◽  
Gene Knockdown ◽  
Naive T Cells ◽  
Naïve T Cells

The differentiation of effector CD8+ T cells is critical for the development of protective responses to pathogens and for effective vaccines. In the first few hours after activation, naive CD8+ T cells initiate a transcriptional program that leads to the formation of effector and memory T cells, but the regulation of this process is poorly understood. Investigating the role of specific transcription factors (TFs) in determining CD8+ effector T-cell fate by gene knockdown with RNAi is challenging because naive T cells are refractory to transduction with viral vectors without extensive ex vivo stimulation, which obscures the earliest events in effector differentiation. To overcome this obstacle, we developed a novel strategy to test the function of genes in naive CD8+ T cells in vivo by creating bone marrow chimera from hematopoietic progenitors transduced with an inducible shRNA construct. Following hematopoietic reconstitution, this approach allowed inducible in vivo gene knockdown in any cell type that developed from this transduced progenitor pool. We demonstrated that lentivirus-transduced progenitor cells could reconstitute normal hematopoiesis and develop into naive CD8+ T cells that were indistinguishable from wild-type naive T cells. This experimental system enabled induction of efficient gene knockdown in vivo without subsequent manipulation. We applied this strategy to show that the TF BATF is essential for initial commitment of naive CD8+ T cells to effector development but becomes dispensable by 72h. This approach makes possible the study of gene function in vivo in unperturbed cells of hematopoietic origin that are refractory to viral transduction.

scholarly journals Freshly Isolated Peyer's Patch, but Not Spleen, Dendritic Cells Produce Interleukin 10 and Induce the Differentiation of T Helper Type 2 Cells

1999 ◽  
Vol 190 (2) ◽  
pp. 229-240 ◽  
Author(s):  
Akiko Iwasaki ◽  
Brian Lee Kelsall
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
T Cell ◽  
Immune Responses ◽  
Ex Vivo ◽  
T Helper ◽  
Phenotypic Analysis ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Ifn Γ

Orally administered antigens often generate immune responses that are distinct from those injected systemically. The role of antigen-presenting cells in determining the type of T helper cell response induced at mucosal versus systemic sites is unclear. Here we examine the phenotypic and functional differences between dendritic cells (DCs) freshly isolated from Peyer's patches (PP) and spleen (SP). Surface phenotypic analysis of CD11c+ DC populations revealed that PP DCs expressed higher levels of major histocompatibility complex class II molecules, but similar levels of costimulatory molecules and adhesion molecules compared with SP DCs. Freshly isolated, flow cytometrically sorted 98–100% pure CD11c+ DC populations from PP and SP were compared for their ability to stimulate naive T cells. First, PP DCs were found to be much more potent in stimulating allogeneic T cell proliferation compared with SP DCs. Second, by using naive T cells from ovalbumin peptide–specific T cell receptor transgenic mice, these ex vivo DCs derived from PP, but not from SP, were found to prime for the production of interleukin (IL)-4 and IL-10 (Th2 cytokines). In addition, PP DCs were found to prime T cells for the production of much lower levels of interferon (IFN)-γ (Th1) compared with SP DCs. The presence of neutralizing antibody against IL-10 in the priming culture dramatically enhanced IFN-γ production by T cells stimulated with PP DCs. Furthermore, stimulation of freshly isolated PP DCs via the CD40 molecule resulted in secretion of high levels of IL-10, whereas the same stimulus induced no IL-10 secretion from SP DCs. These results suggest that DCs residing in different tissues are capable of inducing distinct immune responses and that this may be related to the distinct cytokines produced by the DCs from these tissues.

11-color, 13-parameter flow cytometry: Identification of human naive T cells by phenotype, function, and T-cell receptor diversity

10.1038/84701 ◽  
2001 ◽  
Vol 7 (2) ◽  
pp. 245-248 ◽  
Author(s):  
Stephen C. De Rosa ◽  
Leonard A. Herzenberg ◽  
Leonore A. Herzenberg ◽  
Mario Roederer
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
Cell Receptor ◽  
Naive T Cells ◽  
Naïve T Cells

Human CD62L– memory T cells are less responsive to alloantigen stimulation than CD62L+ naive T cells: potential for adoptive immunotherapy and allodepletion

Blood ◽  
2004 ◽  
Vol 104 (8) ◽  
pp. 2403-2409 ◽  
Author(s):  
Aaron E. Foster ◽  
Marina Marangolo ◽  
Mary M. Sartor ◽  
Stephen I. Alexander ◽  
Min Hu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Memory T Cells ◽  
Donor Cell ◽  
Cell Receptor ◽  
Cell Subpopulation ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Barr Virus

Abstract Selective depletion of alloreactive T cells from allogeneic stem cell grafts can reduce graft-versus-host disease (GVHD) while preserving beneficial effects of T cells including facilitation of engraftment, protection against opportunistic infection, and reduced relapse risk. Memory T cells (CD62L–) represent a population of T cells that have previously encountered pathogens and may contain fewer T cells capable of recognizing neoantigens including recipient allogeneic antigen (aAg). We investigated whether human naive (CD62L+) or memory (CD62L–) T cells had different capacities to respond to aAg by assessing their ability to proliferate in response to and lyse HLA-mismatched Epstein-Barr virus–transformed B cells. Freshly sorted and in vitro expanded CD62L– memory T cells were less responsive to aAg stimulation than were CD62L+ naive T cells but contained higher levels of cytomegalovirus (CMV)–specific T cells. Analysis of T cell receptor (TCR) repertoire showed restricted TCR diversity in the memory T-cell population possibly due to selection associated with chronic exposure to common pathogens. Memory T cells may represent a donor cell subpopulation suitable for enhancing immune reconstitution without increasing the risk of GVHD.

scholarly journals 588 Targeting GITR enhances human tumour-infiltrating T cell functionality in mismatch repair proficient primary colorectal carcinoma and liver metastases

2020 ◽  
Vol 8 (Suppl 3) ◽  
pp. A623-A623
Author(s):  
Yannick Rakké ◽  
Lucia Campos Carrascosa ◽  
Adriaan van Beek ◽  
Valeska de Ruiter ◽  
Michael Doukas ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Colorectal Carcinoma ◽  
Liver Metastases ◽  
Mismatch Repair ◽  
Immune Checkpoint ◽  
Ex Vivo ◽  
Human Tumour ◽  
Functional Assays

BackgroundImmune checkpoint blockade (ICB; e.g. anti-PD-1/-CTLA-4) has been proven to be clinically effective in mismatch repair deficient (dMMR) colorectal carcinoma (CRC). Yet, the majority of patients carry mismatch repair proficient (pMMR) CRC, especially those with liver metastasis, and do not respond to ICB. Here, we studied the effect of immune checkpoint stimulation via GITR targeting on human tumour-infiltrating lymphocyte (TIL) functionality in pMMR primary CRC and liver metastases (CRLM).MethodsHuman TIL were isolated from freshly resected pMMR tumours of patients with primary CRC (stage 1–3) or liver metastases (table 1). GITR expression on TIL was determined using flow cytometry and compared to leukocytes isolated from blood (PBMC) and tumour-free surrounding tissues (tumour-free colon/liver, resp. TFC and TFL). Ex vivo functional assays were used to assess TIL expansion, activation and cytokine/cytotoxic mediator secretion upon CD3/CD28 bead activation and co-stimulation using an antibody-crosslinked recombinant trimeric GITR ligand (GITRL).ResultsGITR was overexpressed on TIL when compared to other stimulatory immune checkpoints (4-1BB, OX40). GITR expression was enhanced on CD4+ and CD8+ TIL compared to PBMC and TFC or TFL compartments in both primary CRC and CRLM. Among CD4+ TIL, GITR was increasingly expressed on CD45RA± FoxP3- helper T (Th), CD45RA- FoxP3int activated helper T (aTh), and CD45RA- FoxP3hi activated regulatory T cells (aTreg), respectively. Within CD8+ TIL, GITR expression was higher on TOX+ PD1Hi and putatively tumour-reactive CD103+ CD39+ TIL.1 Impaired effector cytokine production upon ex vivo PMA/ionomycin stimulation was observed in CD4+ and CD8+ GITR-expressing TIL, hinting to functional exhaustion of the target population. However, recombinant GITRL reinvigorated ex vivo TIL responses by significantly enhancing CD4+ and CD8+ TIL numbers and proinflammatory cytokine secretion in a dose-dependent manner (figure 1). Treg depletion did not fully abrogate the stimulatory effect of GITR ligation on CD4+ and CD8+ T cell expansion, demonstrating that the stimulatory effect was partly exerted via direct targeting GITR on effector T cells. Importantly, GITR-ligation also enhanced expansion of purified CD8+CD39+ TIL. Dual treatment with GITR ligand and nivolumab (anti-PD-1) further enhanced CD8+ TIL responses compared to GITR ligand monotherapy, whereas nivolumab alone did not show any effect.Abstract 588 Table 1Patient characteristicsPatient characteristics of patients included for FACS analysis and/or functional assays. † Pathologic staging was performed according to the AJCC 8th edition criteriaAbstract 588 Figure 1GITR ligation enhances CD4+ and CD8+ TIL expansionTIL were isolated from CRC or CRLM and cultured upon CD3/CD28 activation with or without GITRL (0.1–1.0 ug/mL) for 8 days. TIL numbers were acquired by flow cytometry and normalized to counting beads. Indicated is fold change relative to ctrl-treated TIL (n=10).ConclusionsAgonistic targeting of GITR enhances ex vivo human TIL functionality in pMMR CRC and might therefore be a promising approach for novel mono- or combinatorial immunotherapies in primary CRC and CRLM.AcknowledgementsN/ATrial RegistrationN/AEthics ApprovalThe study was approved by the medical ethics committee of the Erasmus Medical Center (MEC-2012-331).ConsentN/AReferenceDuhen T, Duhen R, Montler R, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat Commun 2018;9(1):2724. doi: 10.1038/s41467-018-05072-0.

scholarly journals Long-Term Depletion of Naive T Cells in Patients Treated for Hodgkin's Disease

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3662-3672 ◽  
Author(s):  
Nobukazu Watanabe ◽  
Stephen C. De Rosa ◽  
Anthony Cmelak ◽  
Richard Hoppe ◽  
Leonore A. Herzenberg ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hodgkin's Disease ◽  
Hodgkin’S Disease ◽  
Cell Subset ◽  
T Cell Subsets ◽  
Naive T Cells ◽  
Naïve T Cells ◽  
Cell Counts

Abstract We investigated the representation of T cells in patients who had been treated for Hodgkin's disease (HD). We found a marked depletion in both CD4 and CD8 naive T-cell counts that persists up to 30 years after completion of treatment. In contrast, CD4 and CD8 memory T-cell subsets recovered to normal or above normal levels by 5 years posttreatment. Thus, the previously-reported long-term deficit in total CD4 T-cell counts after treatment for HD is due to specific depletion of naive T cells. Similarly, total CD8 T-cell counts return to normal by 5 years only because CD8 memory T cells expand to higher than normal levels. These findings suggest that the treatment (mediastinal irradiation) results in a longterm dysregulation of T-cell subset homeostasis. The profound depletion of naive T cells may explain the altered T-cell function in treated patients, including the poor response to immunization after treatment for HD. Further, in some individuals, we identified expansions of unusual subsets expressing low levels of CD8. Eight-color fluorescence-activated cell sorting analyses showed that these cells largely express CD8αα homodimers and CD57, consistent with the phenotype of potentially extrathymically derived T cells. In addition, these cells, both CD4+ and CD4−, are probably cytotoxic lymphocytes, as they express high levels of intracellular perforin. In adults treated for HD, an increased activity of extrathymic T-cell differentiation may partially compensate for the loss of thymic-derived T cells.

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