Efficient lentiviral transduction method to gene modify cord blood CD8+ T cells for cancer therapy applications

Abstract Introduction: Unrelated umbilical cord blood transplantation (UUCBT) is a viable option for those who lack HLA-matched sibling donors. However, opportunistic infections (OI) occurring in the first 100 days, remain the major cause of morbidity and mortality. Viral infections are the primary cause of OI death. As previously shown, cord blood T cells have significantly less preformed effector molecules available intracellularly to kill virally infected cells via the perforin-granzyme pathway than adult PB T cells. Since several virally infected patients control their infections in the absence of specific antiviral therapy ( e.g adenoviral enteris, polyoma cystitis) we postulated that the T cell compartment of those UUCBT recipients who experience early viral infections maty upregulate expression of the perforin exocytosis pathway. In parallel the impact of viral infection on T cell turnover would also be appreciable. Here we report on 19 prospectively studied pediatric patients, all full donor chimera, following myeloablative therapy. Methods: On day +50 we determined by 4-color FACS the expression of intracellular Granzyme, A, B, along with perforin. To monitor T cell turnover proliferating cells were identified by monitoring for the KI-67 nuclear antigen. The expression of the antiapoptosis gene BCL-2 was similarly monitored in both CD4+ and CD8+ T cells. We analyzed their association with the development of de novo OI up to day +100 employing Student’s t-test. Results: Mean age of patients was 6.2 years. 10 of 19 patients developed OI (adenovirus x 4, CMV x 7, EBVx1, parainfluenza x 1) with 5/10 patients experiencing more than one viral infections simultaneously) at a median of 29 days after UUCBT. Of those with OI 6/10 died due to their infections while 8/9 without OI are alive at a median of 15.8 months after UUCBT with one death due to leukemic relapse. Table I presents the correlation between the tested parameters with the development of OI. Patients experiencing viral infections had significantly higher % of their T cells in particular CD8+ T cells equipped with effectors of cytotoxicity and were proliferating in higher percentage compared to those with no active infections. However, the anti-apoptotic protein BCL-2 expression was significantly lower in patients experiencing OI that may lead to their shorter life span and overall T cell lymphopenia observed in OI patietns that we have previously detected in a larger cohort of 102 patients (ASBMT 2004 abstract#48). Conclusion: Correlating with active viral infections significant maturation of cord blood T cells is evident as early as 50 days after UUCBT towards acquiring effector molecules of the perforin pathway. Enhanced T cell proliferation is counteracted by reduced expression of BCL-2 that may lead to the lymphopenia in patients with OI. Future strategies aiming to enhance the longevity of antiviral T cells may protect from death due to viral infections. Univariate analysis VARIABLE MEDIAN VALUE FOR PATIENTS WITH OI MEDIAN VALUE FOR PATIENTS WITHOUT OI t-Test p value % Granzyme A+ T cells 52% 9% 0.006 % Granzyme A+ CD8+ T cells 91% 47% <0.001 % Granzyme B+ T cells 36% 6% 0.036 % Granzyme B+ T cells 87% 39% <0.001 % Perforin+ T cells 38% 4% 0.009 % Perforin+ CD8+ T cells 61% 21% <0.001 % Ki-67+ T cells 27% 16% 0.0041 % Ki-67+ CD8+ T cells 35% 16% 0.0037 BCL-2 expression level (MFI) 87 117 0.028

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Contribution of T-Cell Reconstitution during the Early Phase after Cord Blood Transplantation to Favorable Clinical Outcomes.

Blood ◽

10.1182/blood.v104.11.2227.2227 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2227-2227

Author(s):

Satoshi Takahashi ◽

Nobukazu Watanabe ◽

Jun Ooi ◽

Akira Tomonari ◽

Kashiya Takasugi ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cord Blood ◽

Cd8 T Cells ◽

Early Phase ◽

Cord Blood Transplantation ◽

Interferon Γ ◽

Cell Recovery ◽

Memory Phenotype ◽

Blood Transplantation

Abstract The immaturity of T cells in cord blood is well known in functional assays and phenotypic analyses. During the first several months after cord blood transplantation (CBT), the T cell compartment is recovered by peripheral expansion from those mature and naïve T cells in cord blood grafts and plays an important role in acute graft-versus-host disease (GVHD) and graft-versus-leukemia reaction. Recently, we have reported that adult patients with hematological malignancies receiving CBT from HLA-partially-mismatched unrelated donors (n=68) had a lower risk of severe acute GVHD (> grade II, 7% versus 26%) and transplant-related mortality (9% versus 29% at 1 year) and a higher probability of disease-free survival (74% versus 44% at 2 years) than HLA-matched unrelated bone marrow transplant (BMT) recipients (n=45) in our multivariate analysis (Takahashi et al., Blood, in press). We speculated that the immune reconstitution process over a period of several months after CBT might have contributed to these promising clinical results. Using four-color analysis with CD4, CD8, CD45RA, and CD62L, more than 90% of cord blood CD4+ and CD8+ T cells in the grafts belonged to the naïve fraction. Cytokine expression in cord blood T cells was also suppressed to 0.1% in CD4+ and to 0.9% in CD8+ with positive interferon-γ by intracellular staining, which were significantly lower than those in adult T cells (16.2% in CD4+ and 37.8% in CD8+). Circulating T cell counts normalized after 3 months for CD8+ and 4 months for CD4+ in our CBT recipients, both of which were significantly faster than in previously published studies, which were 9 months for CD8+ and 12 months for CD4+. After T cell recovery, peripheral blood T cells moved from the naïve to the central memory fraction immediately, and then moved to the effector memory fraction. A naïve subset of CD4+ T cells remained (median: 38 cells/μl on day 90, n=12) during the first 3 months, which was significantly higher than in the BMT control (median: 9 cells/μl on day 90, n=5, p=0.015), but showed a low level of CD8+ T cells (median: 14 cells/μl on day 90, n=12), almost the same as in BMT recipients (median: 13 cells/μl on day 90, n=5). Intracellular interferon-γ-producing T cells were detected at 3.4% (0.1–34.2%) in CD4+ and 32.3% (1.1–86.9%) in CD8+ at 1 month post-CBT (n=16), both of which were comparable to post-BMT. To investigate whether these T cells with memory phenotype are functional, we analyzed antigen-specific T-cell recovery using cytomegalovirus (CMV) as a specific antigen. CMV-responsive CD4+ T cells were detected within the first 4 months in all recipients with positive CMV antigenemia (n=13), but CD8+ T cells were detected only in 5 out of 13 cases, probably because of pre-emptive Gancyclovir administration in most antigenemia-positive patients. To conclude, naïve cord blood T cells rapidly increased in number and adopted a memory phenotype showing cytokine-production and antigen-recognition capacity in the early phase after CBT. These data suggest that mature T lymphocytes in cord blood have unique properties and contribute to the favorable clinical outcome of CBT.

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Expansion of Inhibitory NK Receptor (CD94/NKG2A)-Expressing Cytolytic CD8 T Cells and CD4+CD25+ Regulatory T Cells from the Same Cord Blood.

Blood ◽

10.1182/blood.v108.11.1739.1739 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1739-1739

Author(s):

Junji Tanaka ◽

Junichi Sugita ◽

Naoko Kato ◽

Tomomi Toubai ◽

Jun Ibata ◽

...

Keyword(s):

T Cells ◽

Regulatory T Cells ◽

Cord Blood ◽

Cd8 T Cells ◽

Hla Class I ◽

Treg Cells ◽

Class I ◽

Target Cells ◽

Foxp3 Mrna ◽

Effector Cells

Abstract It has recently been shown that inhibitory natural killer cell receptors (NKRs) on not only NK cells but also on T cells negatively regulate NK cell and T cell functions through their binding to MHC class I molecules. The C-type lectin superfamily inhibitory NKR CD94/NKG2A heterodimer recognizes an HLA-E that preferably bound to a peptide derived from the signal sequences of most HLA class I. Therefore, CD94-expressing cells can monitor the global status of HLA class I on the tumor and leukemic cells and induce cytolytic attack without inhibitory signal against HLA class I decreased target cells resulting induction of graft-versus-leukemia (GVL) effect but does not attack normal cells with HLA class I expression resulting no enhancement of graft-versus-host disease (GVHD). On the other hand, CD4+ CD25+ regulatory T cells (Treg) contribute to suppress allogeneic immune responses and prevent transplant rejection and GVHD. In this study, we tried to expand CD94-expressing T cells and Treg cells from the same cord blood cells and then investigated their cytolytic characteristics and immunoregulatory function in order to develop a potential strategy of cell therapy for hematological malignancy. After CD4 enrichment by negative selection using magnetic cell sorting (MACS) (Miltenyi Biotec)(CD4-enriched fraction) from cord blood, CD4+ CD25+ cells were isolated by positive selection with anti-CD25 magnetic microbeads. We could get more than 1,000 fold expansion of CD94-expressing CD8 T cells from CD4-depleted fraction after 8 days culture with immobilized anti-CD3 monoclonal antibody (mAb) (1 μg/mL) and IL-15 (5 ng/mL). Isolated CD4+ CD25+ cells were cultured with anti-CD3/CD28 mAb-coated dynabeads and IL-15 (5 ng/mL) and we could get about 50 fold expansion of Treg cells for 8 days. These expanded Treg cells could suppress allogeneic mixed lymphocyte culture more than 80% (effector cells: Treg cells= 2:1) and expressed FoxP3 mRNA about 100 fold compared with isolated CD25-negative cells. Cytolytic activities of purified CD94-expressing cells (CD94 > 90%) detected by 4 hours 51Cr release assay against K562 were 68.8 ± 16.8 % (n=5). Coculture of CD94-expressing cells with expanded Treg cells (CD94-expressing cell: Treg cells= 1:1, preincubation 4 hours) did not have any effect on cytolytic activities of purified CD94-expressing cells against K562 cells (66.1 ± 19.8 %, n=5). CD94-expressing CD8 T cells with cytolytic activity could be expanded from CD4-deplted fractions and Treg cells with immunosuppressive activity and increased expression level of FoxP3 mRNA could be expanded from CD4-enriched fractions of the same cord blood. Expanded these cytolytic CD94-expressing CD8 cells might be able to induce GVL effect without enhancing GVHD and Treg cells might be able to suppress allogeneic response including GVHD and graft rejection. Therefore, this strategy may be useful to differentiate lymphocytes in cord blood to two different kinds of effector cells exhibiting cytolytic or immunoreguratoly characters.

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Gene expression profiling of CD3γ, δ, ϵ, and ζ chains in CD4+and CD8+T cells from human umbilical cord blood

Hematology ◽

10.1179/102453309x12583347113537 ◽

2010 ◽

Vol 15 (4) ◽

pp. 230-235 ◽

Cited By ~ 6

Author(s):

Shaohua Chen ◽

Lijian Yang ◽

Xiaoye Lu ◽

Bo Li ◽

John Yeuk-Hon Chan ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Cord Blood ◽

Gene Expression Profiling ◽

Umbilical Cord ◽

Cd8 T Cells ◽

Umbilical Cord Blood ◽

Expression Profiling ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord

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Modulation of cord blood CD8+ T-cell effector differentiation by TGF-β1 and 4-1BB costimulation

Blood ◽

10.1182/blood-2003-12-4343 ◽

2005 ◽

Vol 105 (1) ◽

pp. 274-281 ◽

Cited By ~ 22

Author(s):

Young-June Kim ◽

Teresa M. Stringfield ◽

Yan Chen ◽

Hal E. Broxmeyer

Keyword(s):

T Cells ◽

T Cell ◽

Immune Responses ◽

Cord Blood ◽

Cd8 T Cells ◽

Cytotoxic T Cell ◽

Tnf Receptor ◽

Tnf Receptor Family ◽

Tgf Β1 ◽

Receptor Family

Abstract Transforming growth factor-β1 (TGF-β1), an immunosuppressive cytokine, inhibits cytotoxic T cell (CTL) immune responses. In contrast, 4-1BB (CD137), a costimulatory molecule in the tumor necrosis factor (TNF) receptor family, amplifies CTL-mediated antitumor immune responses. We investigated whether TGF-β1 responses could be reversed by 4-1BB costimulation during in vitro differentiation of naive CD8+ T cells into effector CTL cells. TGF-β1 potently suppressed CTL differentiation of human cord blood naive CD8+ T cells as determined by reduced induction of characteristic phenotypes of effector cells and cytotoxic activity. TGF-β1-mediated suppression of CTL differentiation was abrogated by 4-1BB costimulation but not by CD28 or another member in the TNF receptor family, CD30. 4-1BB costimulation suppressed Smad2 phosphorylation induced by TGF-β1, suggesting that 4-1BB effects were at the level of TGF-β1 signaling. 4-1BB effects on the TGF-β1-mediated suppression were enhanced by interleukin 12 (IL-12) but counteracted by IL-4; 4-1BB expression was up- or down-regulated, respectively, by IL-12 and IL-4. IL-4 was more dominant than IL-12 when both cytokines were present during 4-1BB costimulation in the presence of TGF-β1. This indicates critical roles for IL-4 and IL-12 in regulating 4-1BB effects on TGF-β1-mediated suppression. (Blood. 2005;105:274-281)

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Abstract 4080: Expansion of CX3CR1+ cytotoxic CD8+ T cells provides a rationale for IL-15 in combination cancer therapy with PD-1 antibody

10.1158/1538-7445.am2019-4080 ◽

2019 ◽

Author(s):

Henan Zhang ◽

Xin Liu ◽

Siyu Cao ◽

Lingling Chen ◽

Susan Harrington ◽

...

Keyword(s):

T Cells ◽

Cancer Therapy ◽

Cd8 T Cells ◽

Combination Cancer Therapy

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The Feature of δRec-ψJα sjTRECs Level and Frequency of 23 TCR Vβ-Dβ1 sjTRECs in Mononuclear Cells, CD4+ and CD8+ T Cells from Cord Blood and Peripheral Blood of Normal Individuals.

Blood ◽

10.1182/blood.v108.11.3873.3873 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3873-3873

Author(s):

Yangqiu Li ◽

Qingsong Yin ◽

Shaohua Chen ◽

Lijian Yang ◽

Grzegorz Przybylski ◽

...

Keyword(s):

T Cells ◽

Quantitative Analysis ◽

Cord Blood ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Output Function ◽

Cd4 Cells ◽

Cd8 Cells ◽

Normal Individuals

Abstract Thymic recent output function is characterized its importance of thymus to T-cell diversity in the periphery of both children and adults. The generation of TCR diversity occurs in the thymus through recombination of gene segments encoding the variable parts of the TCR α and β chains. During these processes, by-products of the rearrangements are generated in the form of signal joint T-cell receptor excision circles (sjTRECs), which is considered as a very valuable tool to estimate thymic function. Quantitative of δRec-ψJα sjTRECs can direct evaluate the recent thymic output function, but it is unable to analyze the particular thymic output function of different TCR Vβ subfamily naive T cells. The complexity of TCR Vβ repertoire is an important factor for immune reconstitution, quantitative analysis of series TCR Vβ-Dβ sjTRECs could be used to evaluate the levels of different Vβ subfamily naive T cells. In the present study, quantitative analysis of δRec-ψJα sjTRECs was performed in mononuclear cells, CD3+, CD4+ and CD8+T cells from peripheral blood of normal individuals and cord blood by real-time PCR(TaqMan). And the analysis of 23 TCR Vβ-Dβ1 sjTRECs was performed by semi-nested PCR. Different amounts of DNA (corresponding to 2*105, 5*104, 1*104 and 1*103 cells respectively) from all samples were amplified to estimate the frequency of TCR Vβ-Dβ sjTRECs. The mean value of δRec-ψJα sjTRECs was detected in 4.10±3.65/1000 PBMCs, 6.37±5.28/1000 CD3+cells, 3.28±1.24/1000 CD4+cells, 4.67±3.63/1000 CD8+cells from normal individuals (n=14) and 35.59±47.56/1000 CBMC, 71.48±86.42/1000 CD3+cells, 41.02±32.9/1000 CD4+ cells, 52.05±52.32/1000 CD8+cells from cord blood (n=9) (p=0.0208, p=0.0096, p=0.0003, p=0.0026, respectively). A part of Vβ subfamily sjTRECs could be detected in all samples from cord blood (Vβ2, 3, 4, 5, 10, 13, 14, 15, 19 and 22) and peripheral blood (Vβ10, 13 and 14) at 5*104 cells level, some of Vβ subfamily sjTRECs could be detected in 1*103 cells level. The frequencies of 23 Vβ-Dβ1 sjTRECs were different at the same cellular concentration. The number of detectable Vβ subfamily sjTRECs was 22.00±0.94/2×105, 18.8±1.87/5×104, 10.40±2.99/1×104 and 0.78±1.39/1×103 CBMCs, as compared with 18.70±2.45/2×105 (p=0.002), 13.7±2.67/5×104 (p<0.001), 5.5±2.07/1×104 (p=0.001) and 0.50±0.71/1×103 (p=0.739) in PBMCs from normal individuals. Similar results were found in CD4+ and CD8+ T cells which were sorted from both CBMCs and PBMCs, the number of detectable Vβ subfamily sjTRECs was 13.90±2.38/1×104 CD4+cells, 11.5±1.96/1×104CD8+cells from cord blood and 5.6±2.68/1×104 CD4+cells (p<0.001) and 8.2±2.57/1×104CD8+cells (p>0.005) from normal individuals. The results indicate that the number of detectable sjTRECs of Vβ subfamilies and the frequencies of most Vβ-Dβ1 sjTRECs in normal PBMCs, CD4+ and CD8+T cells were obviously lower than those in cord blood. In conclusions, the results provide the base data of naïve T cells levels and thymic recent output function in cord blood and peripheral blood of normail individuals in chinese.

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Higher HHV-6 Reactivation After Cord Blood Allo-SCT Is Not Related to HHV-6 Cell Receptor CD46 Expression

Blood ◽

10.1182/blood.v120.21.1931.1931 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1931-1931

Author(s):

Patrice Chevallier ◽

Nelly Robillard ◽

Marina Illiaquer ◽

Julie Esbelin ◽

Mohamad Mohty ◽

...

Keyword(s):

Stem Cells ◽

T Cells ◽

B Cells ◽

Cord Blood ◽

Nk Cells ◽

B Lymphocytes ◽

Cd8 T Cells ◽

Peripheral Blood ◽

Cd4 T Cells ◽

Cell Receptor

Abstract Abstract 1931 Introduction: Cord Blood (CB) are increasingly used as an alternative stem cells source in adults for allogeneic Stem Cell Transplantation (allo-SCT). The risk of human herpes virus (HHV-6) reactivation is significantly higher after CB transplant vs unrelated peripheral blood stem cells (PBSC) allo-SCT (Chevallier et al, BMT 2010). Higher HHV-6 cell receptor CD46 expression on progenitor cells in CB may explain this difference (Thulke et al, Virol J 2006). Patients and Methods: We have prospectively compared the HHV-6 cell receptor CD46 expression on various cell subsets of three freshly harvested blood sources on one hand and of three graft sources on the other hand. 52 samples were used for the purpose of this study. They were issued from peripheral blood (PB, n=10), G-CSF mobilised PB (GCSF-PB, n=10), cord blood (CB, n=10), unmanipulated bone marrow (uBM, n=5), leukapheresis product (LP, n=10) and thawed CB graft (n=7). CD46 expression was assessed by FACS analysis using a FACS CANTO II (BD Biosciences, San Jose, CA, USA) on total lymphocytes, monocytes, NK cells, T and B cells subsets, plasmacytoid (pDCs) dendritic cells and stem cells. Results: As all cell subsets were found CD46 positive, CD46 mean fluorescence intensity (MFI) was then considered for comparison. When considering the three blood sources, CD46 MFI were found similar on T cells, CD4-/CD8+ and CD4-/CD8- T cells, NKT cells, Tregs, memory B lymphocytes, pDCs and CD34+ stem cells. CD46 MFI was significantly lower on CD4+/CD8- and CD4+/CD8+ T cells, transitional B cells, total and naïve B lymphocytes, and NK cells in CB while higher on monocytes. The highest CD46 MFI was observed on monocytes in CB and on CD4+/CD8+ T cells in GCSF-PB and PB. Also, highest CD46 MFI was detected on T cells compared to B lymphocytes and NK cells in all blood sources while CD46 MFI was higher on CD4+/CD8- T cells compared to CD8+/CD4- T cells. When considering the three graft sources, CD46 MFI was similar on CD4-/CD8- T cells and NKT cells. CD46 MFI was found significantly lower on all other sub-populations in thawed CB graft, except monocytes. The highest CD46 MFI was observed on monocytes in CB graft, on CD4+/CD8+ T cells in LP and on monocytes and on CD4+/CD8- T cells in uBM. Also, highest CD46 MFI was detected on T cells compared to B lymphocytes and NK cells in all graft sources while CD46 MFI was higher on CD4+/CD8- T cells compared to CD8+/CD4- T cells. Conclusion: This original study shows strong differences in term of quantitative CD46 expression between several blood and grafts samples. Our results suggest that other factors (such as another HHV-6 cell surface receptor) than the qualitative CD46 expression play a role in the higher HHV-6 reactivation observed after CB transplant in adults. Disclosures: No relevant conflicts of interest to declare.

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