Lymphocyte Co-Expression of CD38/HLA-DR Is a Marker for Anti-Tumor Activity in Hodgkin Lymphoma in Vitro: Evidence for a PD1-Independent Mechanism

Abstract Introduction The immunosuppressive tumor microenvironment has become of increasing interest in Hodgkin lymphoma (HL), in particular due to the recent success of checkpoint inhibitors (CI) as part of therapeutic strategies. The mechanism of these agents action in HL, however, remains elusive. Some studies have shown that cytotoxic T-lymphocytes may not be responsible for clinical efficacy, and that tumor-associated macrophages may also be targeted by these agents. We recently described a positive association between increasing proportion of CD38/HLA-DR co-positive lymphocyte in affected nodal tissue and clinical outcome in children with HL. We developed an in vitro model to further evaluate our findings, in this study. Methods Peripheral blood mononuclear cells collected from healthy volunteers were used to generate effector cytoxic T lymphocytes (CTL), natural killer (NK) and CD4 positive T (CD4+T) cells by incubating with IL-15 and IL-2. Both a short-term (4-day) incubation and a longer incubation was used to generate lymphocytes with low-level and higher CD38/HLA-DR co-positive cells, respectively. CD3/CD8 co-positive CTL, NK (CD56-positive/CD3-negative) and CD4-positive T cells were isolated using MACS system. In addition to CD38/HLA-DR expression, isolated cells were evaluated for expression of CD279 (PD-1) and CD274 (PDL-1) by flow cytometry. Two HL cell lines, HDLM-2 (nodular sclerosis HL) and KMH-2 (mixed cellular HL) were used as targets in effector cell-mediated cytotoxicity experiments. Cells were incubated at various effector:target ratios, and HL cell death was measured with a flow cytometric cell-mediated cytotoxicity assay. The resuts were corrected for alloreactive cell elimination. Blocking antibodies against PD-1 and PDL-1 were used for cytoxicity experiments, using CTL and CD4-positive T cells as effector cells, as well. Results Higher CD38/HLA-DR co-expression was seen in CTL after longer incubation (day 11) with IL-2 and IL-15, while peak expression was reached earlier in NK cells (day 4). Both CTL and NK cells demonstrate cytotoxicity against HDLM-2 and KMH-2 cell lines. Cytotoxicity was increased, as evidenced by lower (lethal unit 20%) LU20 effector:target ratio levels, at day 11 of incubation (compared to day 5) for CTL for both cell lines: 0.5 (day 5) vs 0.29 (day 11) for KMH-2 (p=0.02) and 1.1 (day 5) vs. 0.4 (day 11) for HDLM-2 (p=0.15) cells. There was no difference between cytotoxicity with CTL compared to NK cells for either cell line at day 11. CTL, NK cells, and CD4-positive T cells all expressed both PD-1 and PDL-1, with no difference between cell types in percent positivity or mean channel intensity after cytokine incubation. PD-1 expression increased with incubation time in CTL, peaking at 50.5% on day 10, as opposed to NK cells, where it peaked at day 5-7. The co-expression of CD38/HLA-DR was higher in CTL compared to CD4-positive T cells (79% vs. 37% at day 7). PD-1 blockade did not inhibit CTL or CD4-positive T cell-mediated cytotoxicity in either cell line. This was also the case after PDL-1 blockade on tumor cells, indicating PD-1/PDL-1 pathway-independent HL cell elimination by CD38/HLA-DR co-positive CTL and CD4-positive T cells. Discussion Our results indicate that higher CD38/HLA-DR co-expression in CTL was associated with superior elimination of HL cells in vitro supporting our recent in vivo findings. Induced co-expression of CD38/HLA-DR was higher in CTL compared with NK cells and reached a peak level earlier in NK cells. Increasing expression of PD-1 and PDL-1 was observed for all three effectors cells with longer incubation time. Interestingly, there was no change in CTL or CD4-positive T cell-mediated cytoxicity of HL cells following PD-1 and PDL-1 blockade in vitro. In conclusion, both CTL and NK cells are effective against HL cells. The anti-tumor activity of CTL correlated with increasing levels of CD38/HLA-DR expression in our experimental model. Cytotoxicity was enhanced despite increased expression of PD-1, and, therefore, appears to be independent of the PD-1/PDL-1 pathway, suggesting involvement of other operational mechanisms. This model could be useful in further elucidating the interactions between immune effectors and HL cells. Disclosures No relevant conflicts of interest to declare.

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A Novel Approach for Treatment of T-Cell Malignancies: Targeting T-Cell Receptor Vβ Families

Blood ◽

10.1182/blood-2020-142010 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 28-29

Author(s):

Jie Wang ◽

Katarzyna Urbanska ◽

Prannda Sharma ◽

Mathilde Poussin ◽

Reza Nejati ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Cell Receptor ◽

Brentuximab Vedotin ◽

T Cell Lymphomas ◽

T Cell Malignancies

Background: Peripheral T-cell lymphomas (PTCL) encompass a highly heterogeneous group of T-cell malignancies and are generally associated with a poor prognosis. Combination chemotherapy results in consistently poorer outcomes for T-cell lymphomas compared with B-cell lymphomas.1 There is an urgent clinical need to develop novel approaches to treatment of PTCL. While CD19- and CD20-directed immunotherapies have been successful in the treatment of B-cell malignancies, T-cell malignancies lack suitable immunotherapeutic targets. Brentuximab Vedotin, a CD30 antibody-drug conjugate, is not applicable to PTCL subtypes which do not express CD30.2 Broadly targeting pan-T cell markers is predicted to result in extensive T-cell depletion and clinically significant immune deficiency; therefore, a more tumor-specific antigen that primarily targets the malignant T-cell clone is needed. We reasoned that since malignant T cells are clonal and express the same T-cell receptor (TCR) in a given patient, and since the TCR β chain in human α/β TCRs can be grouped into 24 functional Vβ families targetable by monoclonal antibodies, immunotherapeutic targeting of TCR Vβ families would be an attractive strategy for the treatment of T-cell malignancies. Methods: We developed a flexible approach for targeting TCR Vβ families by engineering T cells to express a CD64 chimeric immune receptor (CD64-CIR), comprising a CD3ζ T cell signaling endodomain, CD28 costimulatory domain, and the high-affinity Fc gamma receptor I, CD64. T cells expressing CD64-CIR are predicted to be directed to tumor cells by Vβ-specific monoclonal antibodies that target tumor cell TCR, leading to T cell activation and induction of tumor cell death by T cell-mediated cytotoxicity. Results: This concept was first evaluated in vitro using cell lines. SupT1 T-cell lymphoblasts, which do not express a native functioning TCR, were stably transduced to express a Vβ12+ MART-1 specific TCR, resulting in a Vβ12 TCR expressing target T cell line.3 Vβ family specific cytolysis was confirmed by chromium release assays using co-culture of CD64 CIR transduced T cells with the engineered SupT1-Vβ12 cell line in the presence of Vβ12 monoclonal antibody. Percent specific lysis was calculated as (experimental - spontaneous lysis / maximal - spontaneous lysis) x 100. Controls using no antibody, Vβ8 antibody, and untransduced T cells did not show significant cytolysis (figure A). Next, the Jurkat T cell leukemic cell line, which expresses a native Vβ8 TCR, was used as targets in co-culture. Again, Vβ family target specific cytolysis was achieved in the presence of CD64 CIR T cells and Vβ8, but not Vβ12 control antibody. Having demonstrated Vβ family specific cytolysis in vitro using target T cell lines, we next evaluated TCR Vβ family targeting in vivo. Immunodeficient mice were injected with SupT1-Vβ12 or Jurkat T cells with the appropriate targeting Vβ antibody, and either CD64 CIR T cells or control untransduced T cells. The cell lines were transfected with firefly luciferase and tumor growth was measured by bioluminescence. The CD64 CIR T cells, but not untransduced T cells, in conjunction with the appropriate Vβ antibody, successfully controlled tumor growth (figure B). Our results provide proof-of-concept that TCR Vβ family specific T cell-mediated cytolysis is feasible, and informs the development of novel immunotherapies that target TCR Vβ families in T-cell malignancies. Unlike approaches that target pan-T cell antigens, this approach is not expected to cause substantial immune deficiency and could lead to a significant advance in the treatment of T-cell malignancies including PTCL. References 1. Coiffier B, Brousse N, Peuchmaur M, et al. Peripheral T-cell lymphomas have a worse prognosis than B-cell lymphomas: a prospective study of 361 immunophenotyped patients treated with the LNH-84 regimen. The GELA (Groupe d'Etude des Lymphomes Agressives). Ann Oncol Off J Eur Soc Med Oncol. 1990;1(1):45-50. 2. Horwitz SM, Advani RH, Bartlett NL, et al. Objective responses in relapsed T-cell lymphomas with single agent brentuximab vedotin. Blood. 2014;123(20):3095-3100. 3. Hughes MS, Yu YYL, Dudley ME, et al. Transfer of a TCR Gene Derived from a Patient with a Marked Antitumor Response Conveys Highly Active T-Cell Effector Functions. Hum Gene Ther. 2005;16(4):457-472. Figure Disclosures Schuster: Novartis, Genentech, Inc./ F. Hoffmann-La Roche: Research Funding; AlloGene, AstraZeneca, BeiGene, Genentech, Inc./ F. Hoffmann-La Roche, Juno/Celgene, Loxo Oncology, Nordic Nanovector, Novartis, Tessa Therapeutics: Consultancy, Honoraria.

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Dysregulation of Nuclear Factor of Activated T-Cells (NFAT1) in Human T-Cell Acute Lymphocytic Leukemia (T-ALL): Possible Role of Inactivated NFAT1 in Leukemogenesis.

Blood ◽

10.1182/blood.v108.11.1849.1849 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1849-1849

Author(s):

M. Kozik ◽

M.L. Lesniewski ◽

Y. Hegerfeldt ◽

R.R. Brewka ◽

L.R. Fanning ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Western Blot ◽

Cell Line ◽

Cell Lines ◽

Surface Marker ◽

Apoptotic Cells ◽

Hla Dr ◽

Surface Marker Analysis

Abstract Introduction: NFAT1 is a transcription factor integral for the regulation of T-cell proliferation, differentiation, and apoptosis. NFAT1 is present in the cytoplasm of resting T-cells and upon stimulation (ionomycin, antigen or anti-CD3) is dephosphorylated via calcineurin and translocates to the nucleus to associate with other transcription factors (AP1). We sought to determine whether NFAT1 regulation in T-cells contributes to leukemogenesis. Initial in vitro analyses incorporated several T-ALL cell lines all derived from the peripheral blood of patients including 3 mature T-cell lines: CCRF-CEM (no clear chromosomal abnormalities), Jurkat (karyotype 46, XY, −2, −18, del(2) (p21p23), del(18) (p11.2)) Loucy (translocation t(16;20)(p12;q13), and p53 overexpression), and one immature T-cell line Molt-4 (hypertetraploid chromosomes and 6q-, t(7;7)) (ATCC Manassas, VA). Methods: T-ALL cell lines were cultured in accordance with ATCC guidelines. Surface marker analysis for CD34, CD38, HLA-DR, CD3, CD4, CD8, CD2, and CD7 was performed (BD Biosciences). Cytoplasmic and nuclear extracts were prepared from cell lines and control adult blood (AB) CD3+ cells. Cell lysates (20μg) were examined by Western blot. Blots were probed with anti-NFAT1 antibody (BD Biosciences) and protein loading controls β2-microglobulin (Abcam) and lamin A/C (Santa Cruz Biotechnology). Bands were visualized using Supersignal West Pico chemiluminescent substrate (Pierce) and quantitated using NIH imageJ software. Apoptotic assays were performed by treating samples with 0.832 μM cyclosporin A (CsA) for 48 hrs, then determining the percentage of early apoptotic cells using an Annexin V/Propidium iodide flow cytometric kit (Calbiochem). Results: Surface marker analysis confirmed that Loucy, Jurkat, and CCRF-CEM cells possess a mature phenotype, whereas Molt-4 cells possess a more immature phenotype (Table 1). Western blot analysis showed Loucy cells were unique in that NFAT1 was absent in the nuclear fraction, indicating NFAT1 is only present in it’s inactive form in this cell line. Among the T-ALL cell lines, only Loucy showed no difference in the proportion of apoptotic cells following CsA treatment. Table 1. Phenotype Analysis of T-ALL Cell Lines Surface Antigens CCRF-CEM JURKAT LOUCY MOLT-4 Percentage of Total Cells CD2 83.97 96.16 4.96 91.53 CD7 99.74 87.84 99.96 57.88 CD3 18.22 52.15 96.48 8.33 CD4 62.42 54.56 12.62 42.64 CD8 97.42 5.32 0.81 83.19 CD34 14.80 2.62 0.65 55.78 CD38 93.59 89.40 92.92 99.66 HLA-DR 2.31 2.62 0.56 0.33 Conclusions: Despite Loucy cells’ mature phenotype, they exhibited distinct NFAT1 dysregulation compared to other mature cell lines. CsA treatment of Loucy cells failed to effect NFAT1 translocation to the nucleus and induction of apoptosis, as NFAT1 remained in the cytoplasm. Loucy’s insensitivity to CsA with regards to apoptosis signaling supports the idea for a role for NFAT1 in Ca(2+) signaling cascade for apoptosis in T-cells. These results also suggest a possible role of cytosolic NFAT1 in leukemiogenesis, since the amount of protein seen in whole cell extracts between the various cell lines was nearly the same. The translocation in Loucy cells is in the same chromosomal location as the NFAT1 gene locus. Studies are ongoing to understand the role of NFAT1 in T-ALL initiation and progression.

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Ex vivo isolation of RCC-reactive CD8+ CTL clones from HLA-identical allogeneic donor T cell lines stimulated by CD80-transfected tumor cells

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.15625 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 15625-15625 ◽

Cited By ~ 1

Author(s):

S. S. Tykodi ◽

J. A. Thompson ◽

B. M. Sandmaier ◽

M. B. Maris ◽

R. Storb ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Lines ◽

Ex Vivo ◽

Unrelated Donor ◽

T Cell Antigens ◽

Ifn Γ ◽

T Cell Lines ◽

Anti Tumor Activity

15625 Background: Regression of metastatic renal cell carcinoma (mRCC) is observed in a minority of patients treated by immunotherapies such as interleukin-2 (IL-2), interferon-a (IFN-a), or reduced-intensity allogeneic hematopoietic cell transplantation. However, the development of specific cellular immunotherapies for mRCC has been hindered by the lack of molecularly characterized T cell antigens with preferential expression on RCC cells. We have developed an ex vivo strategy for the isolation of RCC-reactive CD8+ CTL clones that may facilitate the identification of novel RCC-associated T cell antigens. Methods: RCC tumor lines were established from two patients with mRCC presenting to our institution for allogeneic HCT received from either an HLA-matched sibling or volunteer unrelated donor. Irradiated RCC tumor lines that were unmodified or transfected with a cDNA for human CD80 were used to stimulate responder CD8+ T cells isolated from pretransplant patient (autologous) or donor-derived (allogeneic) blood samples in mixed lymphocyte/tumor cell (MLTC) cultures supplemented with recombinant human IL-7 and IL-12 (stimulation #1) or IL-2 (2nd and subsequent stimulations). T cell lines with anti-tumor activity measured by IFN-γ ELISA were then cloned by limiting dilution. Results: After two or more in vitro stimulations, allogeneic CD8+ T cell lines stimulated by CD80- transfected RCC tumor cells, but not the other MLTC culture combinations tested demonstrated tumor-specific IFN-γ release. CD3+/CD8+/TCRaβ+ CTL clones with potent in vitro anti-tumor activity for unmodified RCC tumor were isolated from both sibling- and unrelated- donor derived T cell lines. Three such clones with unique specificities for allogeneic targets recognized the unmodified RCC tumor but not LCL or fibroblast target cells isolated from the same patient suggesting tumor-restricted expression of the target antigens. Conclusions: Ex vivo MLTC culture utilizing CD80-transfected RCC tumor and HLA- matched allogeneic responder CD8+ T cells warrants further study as a strategy to isolate CTL clones that may be used to identify novel RCC-associated T cell antigens. No significant financial relationships to disclose.

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The influence of methylprednisolone on the ability of CD4+CD95+HLA-DR+ T-cells to produce proinflammatory medators in cultures of TCR-activated CD3+CD45RO+ T-lymphocytes from patients with rheumatoid arthritis

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20176303255 ◽

2017 ◽

Vol 63 (3) ◽

pp. 255-265

Author(s):

N.M. Todosenko ◽

O.G. Khaziakhmatova ◽

K.A. Yurova ◽

I.P. Malinina ◽

L.S. Litvinova

Keyword(s):

Rheumatoid Arthritis ◽

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Control Group ◽

Proinflammatory Mediators ◽

Hla Dr ◽

Healthy Donors ◽

Autoreactive Cells

The effect of different concentrations of the glucocorticoid (GC) methylprednisolone (MP) on CD4+CD95+HLA-DR+ T-cells and their ability to produce proinflammatory mediators in cultures of TCR-stimulated CD3+CD45RO+ T-lymphocytes in the in vitro system was investigated. T cells were obtained from healthy donors and patients with rheumatoid arthritis (RA).Under conditions of TCR-activation, MP increased the number of CD4+HLA-DR+CD95+ cells in CD3+CD45RO+ cultures obtained from RA patients and did not change their content in the control group. In general, MP decreased production of proinflammatory factors (IFN-, IL-2, IL-17, IL-21 and TNF-) by TCR-activated CD3+CD45RO+ cells from healthy donors and RA, consistent with the overall immunosuppressive mechanism of GC action. The correlation between CD4+CD45RO+HLA-DR+CD95+ T-cell contents and parameters reflecting production of proinflammatory mediators (IL-17, IL-21 and TNF-) in RA patients indicates maintenance of the pro-inflammatory potential of this T-cell population exposed to GC action. We suggest that relative resistance of CD4+CD45RO+CD95+HLA-DR+ T-cells of RA patients to the suppressor effect of GC leads to maintenance and even enhancement in the functional capacities of autoreactive cells in the pathogenesis of RA.

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A Bispecific Antibody Targeting CD117 and CD3 Enables T Cell Mediated Killing of CD117-Expressing Healthy and Malignant Hematopoietic Cells

Blood ◽

10.1182/blood-2021-147676 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2354-2354

Author(s):

Jonathan D Kiefer ◽

Renier Myburgh ◽

Norman F Russkamp ◽

Laura Volta ◽

Adrian Guggisberg ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Cell Line ◽

Cell Lines ◽

Target Cell ◽

Target Cells ◽

Human T Cells ◽

Blast Cells ◽

University Of Zurich

Abstract INTRODUCTION: Hematopoietic stem and progenitor cells (HSPCs) support life-long hematopoiesis. A single HSPC can also be at the origin of hematological malignancies, such as Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS). Allogeneic HSCT with the intent to eliminate recipient AML or MDS and at the same time replace recipient HSPC with donor-HSPC and immune cells is a life-saving therapeutic option for many patients. However, chemotherapy (and sometimes in addition gamma-irradiation based conditioning regiments) prior to HSCT are associated with substantial toxicity. Thus, due to benefit-outweighing treatment-related toxicity and mortality, frail, multi-morbid and elderly patients are usually excluded from potentially curative allo-HSCT approaches. For these reasons, more selective preconditioning strategies, leading to residual AML/MDS elimination and creating "space" for incoming HSPCs, are required. Selective targeting of CD117 with monoclonal antibodies has been proposed as a strategy to remove endogenous HSPCs, enabling an effective but mild preconditioning. However, specific conditioning of AML and MDS patients, prior to HSCT, might require a more potent effector cell type. We hypothesized that a CD117 and CD3 binding, T cell engaging and activating antibody construct (CD117xCD3 TEA) with a short half-life might be an ideal means to selectively eliminate CD117-expressing healthy HSPCs and residual CD117-expressing AML or MDS cells prior to allo-HSCT. METHODS: We cloned and expressed CD117xCD3 TEA in tandem scFv format and produced it by transient gene expression in Chinese hamster ovary cells (CHO-S). The fusion proteins were purified to homogeneity by protein A affinity chromatography. We derived target cell lines with varying surface levels of CD117 (high, medium and low) from CD117 negative parental cell lines HL-60 and MOLM-14 (Myburgh et al., Leukemia, 2020). To assess T cell mediated killing of target cells, we mixed them with human T cells (purified and enriched after negative selection) at varying Effector-to-Target (E:T) cell ratios and added CD117xCD3 TEA at different concentrations. The mixture was incubated and specific killing was quantified via flow cytometry at different time-points. RESULTS: In order to characterize the biocidal properties of CD117xCD3 TEA, we performed in vitro killing experiments against cell lines, HSCPs from healthy donors and blast cells from AML patients. A dose-dependent in vitro killing of the cell lines was observed in the presence of various concentrations of CD117xCD3 TEA and of human T cells at an E:T cell ratio of 10:1 after 24h. The HL60 CD117 high cell line was efficiently lysed (~90%) at 100 ng/ml of CD117xCD3 TEA, corresponding to ~1.8 nM. In similar experiments with different E:T cell ratios, we observed that both HL60 CD117 high and CD117 medium cells could be quantitatively killed at E:T ratios as low as 1:1, while the killing of CD117 low cells required a higher density of T cells. The biocidal effect on non-transduced HL60 cells was negligibly low, confirming the requirement of a simultaneous engagement of CD117 and CD3 for specific killing. We repeated the same experiment with an engineered MOLM14 cell line, which also expressed CD117 at comparable high levels, incubating the target cell line with human T cells at an E:T of 1:1 for 24, 48 or 72, 120 or 192 hours. Complete killing of the target cell line was achieved at 120 and 192 hours and after supplemental addition of T cells and CD117xCD3 TEA at 72 hours (see example figure). Experiments with primary cells (HSPCs from healthy donors or blast cells from AML patients) at an E:T of 1:1 confirmed specific killing of target cells in an antigen-density- and concentration-dependent manner after 48h. CONCLUSIONS: We have generated a novel bispecific antibody, which binds to human CD117 (expressed on HSCPs and AML/MDS blast cells) and to CD3 (expressed on T cells), which we term CD117xCD3 TEA. The antibody induces selective T cell-mediated killing of cell lines with different surface levels of CD117, as well as of healthy HSPCs and primary human AML cells. Thus, the newly generated CD117xCD3 TEA might be developed clinically in order to erradicate residual AML/MDS and at the same time serve as a milder preconditioning approach prior to allo-HSCT in frail AML/MDS patients. Figure 1 Figure 1. Disclosures Kiefer: ETH Zurich: Current Employment, Patents & Royalties: CD117xCD3 TEA. Myburgh: University of Zurich: Patents & Royalties: CD117xCD3 TEA. Guggisberg: F. Hoffmann-La Roche AG: Current Employment. Abdelmotaleb: F. Hoffmann-La Roche AG: Current Employment. Mock: Philogen S.p.A.: Current Employment. Neri: Philogen S.p.A.: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: Multiple patents on vascular targeting; ETH Zurich: Patents & Royalties: CD117xCD3 TEA. Manz: University of Zurich: Patents & Royalties: CD117xCD3 TEA; CDR-Life Inc: Consultancy, Current holder of stock options in a privately-held company.

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Quantitative assessment of the pool size and subset distribution of cytolytic T lymphocytes within human resting or alloactivated peripheral blood T cell populations.

Journal of Experimental Medicine ◽

10.1084/jem.158.2.571 ◽

1983 ◽

Vol 158 (2) ◽

pp. 571-585 ◽

Cited By ~ 84

Author(s):

A Moretta ◽

G Pantaleo ◽

L Moretta ◽

M C Mingari ◽

J C Cerottini

Keyword(s):

Monoclonal Antibody ◽

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Peripheral Blood ◽

Great Majority ◽

T Cell Subsets ◽

Cytolytic T Lymphocytes ◽

Subset Distribution

In order to directly assess the distribution of cytolytic T lymphocytes (CTL) and their precursors (CTL-P) in the two major subsets of human T cells, we have used limiting dilution microculture systems to determine their frequencies. The two subsets were defined according to their reactivity (or lack thereof) with B9.4 monoclonal antibody (the specificity of which is similar, if not identical, to that of Leu 2b monoclonal antibody). Both B9+ and B9- cells obtained by sorting peripheral blood resting T cells using the fluorescence-activated cell sorter (FACS) were assayed for total CTL-P frequencies in a microculture system that allows clonal growth of every T cell. As assessed by a lectin-dependent assay, approximately 30% of peripheral blood T cells were CTP-P. In the B9+ subset (which represents 20-30% of all T cells), the CTL-P frequency was close to 100%, whereas the B9- subset had a 25-fold lower CTL-P frequency. It is thus evident that 90% and 10% of the total CTL-P in peripheral blood are confined to the B9+ or B9- T cell subsets, respectively. Analysis of the subset distribution of CTL-P directed against a given set of alloantigens confirmed these findings. CTL-P frequencies were also determined in B9+ and B9- subsets derived from T cells that had been activated in allogenic mixed leucocyte cultures (MLC). Approximately 10% of MLC T cells were CTL-P. This frequency was increased 3.5-fold in the B9+ subset, whereas the B9- subset contained only a small, although detectable number of CTL-P. Moreover, the great majority of the (operationally defined) CTL-P in MLC T cell population were found to be directed against the stimulating alloantigens, thus indicating a dramatic increase in specific CTL-P frequencies following in vitro stimulation in bulk cultures.

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DNAM-1 promotes activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells and tumors

Journal of Experimental Medicine ◽

10.1084/jem.20081752 ◽

2008 ◽

Vol 205 (13) ◽

pp. 2965-2973 ◽

Cited By ~ 195

Author(s):

Susan Gilfillan ◽

Christopher J. Chan ◽

Marina Cella ◽

Nicole M. Haynes ◽

Aaron S. Rapaport ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Nk Cells ◽

Adhesion Molecules ◽

Cd8 T Cells ◽

Nk Cell ◽

Cd8 T Cell ◽

Antigen Presenting

Natural killer (NK) cells and CD8 T cells require adhesion molecules for migration, activation, expansion, differentiation, and effector functions. DNAX accessory molecule 1 (DNAM-1), an adhesion molecule belonging to the immunoglobulin superfamily, promotes many of these functions in vitro. However, because NK cells and CD8 T cells express multiple adhesion molecules, it is unclear whether DNAM-1 has a unique function or is effectively redundant in vivo. To address this question, we generated mice lacking DNAM-1 and evaluated DNAM-1–deficient CD8 T cell and NK cell function in vitro and in vivo. Our results demonstrate that CD8 T cells require DNAM-1 for co-stimulation when recognizing antigen presented by nonprofessional antigen-presenting cells; in contrast, DNAM-1 is dispensable when dendritic cells present the antigen. Similarly, NK cells require DNAM-1 for the elimination of tumor cells that are comparatively resistant to NK cell–mediated cytotoxicity caused by the paucity of other NK cell–activating ligands. We conclude that DNAM-1 serves to extend the range of target cells that can activate CD8 T cell and NK cells and, hence, may be essential for immunosurveillance against tumors and/or viruses that evade recognition by other activating or accessory molecules.

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Epiligrin, a component of epithelial basement membranes, is an adhesive ligand for alpha 3 beta 1 positive T lymphocytes.

The Journal of Cell Biology ◽

10.1083/jcb.121.5.1141 ◽

1993 ◽

Vol 121 (5) ◽

pp. 1141-1152 ◽

Cited By ~ 126

Author(s):

E A Wayner ◽

S G Gil ◽

G F Murphy ◽

M S Wilke ◽

W G Carter

Keyword(s):

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Basement Membrane ◽

B Cell ◽

Cell Lines ◽

B Cell Lymphoma ◽

Lymphokine Activated Killer Cells ◽

Cutaneous Inflammation ◽

Epidermal Basement Membrane

The cutaneous T cell lymphomas (CTCL), typified by mycosis fungoides, and several chronic T cell mediated dermatoses are characterized by the migration of T lymphocytes into the epidermis (epidermotropism). Alternatively, other types of cutaneous inflammation (malignant cutaneous B cell lymphoma, CBCL, or lymphocytoma cutis, non-malignant T or B cell type) do not show evidence of epidermotropism. This suggests that certain T lymphocyte subpopulations are able to interact with and penetrate the epidermal basement membrane. We show here that T lymphocytes derived from patients with CTCL (HUT 78 or HUT 102 cells), adhere to the detergent-insoluble extracellular matrix prepared from cultured basal keratinocytes (HFK ECM). HUT cell adhesion to HFK ECM was inhibitable with monoclonal antibodies (mAbs) directed to the alpha 3 (P1B5) or beta 1 (P4C10) integrin receptors, and could be up-regulated by an activating anti-beta 1 mAb (P4G11). An inhibitory mAb, P3H9-2, raised against keratinocytes identified epiligrin as the ligand for alpha 3 beta 1 positive T cells in HFK ECM. Interestingly, two lymphocyte populations could be clearly distinguished relative to expression of alpha 3 beta 1 by flow cytometry analysis. Lymphokine activated killer cells, alloreactive cytotoxic T cells and T cells derived from patients with CTCL expressed high levels of alpha 3 beta 1 (alpha 3 beta 1high). Non-adherent peripheral blood mononuclear cells, acute T or B lymphocytic leukemias, or non-cutaneous T or B lymphocyte cell lines expressed low levels of alpha 3 beta 1 (alpha 3 beta 1low). Resting PBL or alpha 3 beta 1low T or B cell lines did not adhere to HFK ECM or purified epiligrin. However, adhesion to epiligrin could be up-regulated by mAbs which activate the beta 1 subunit indicating that alpha 3 beta 1 activity is a function of expression and affinity. In skin derived from patients with graft-vs.-host (GVH) disease, experimentally induced delayed hypersensitivity reactions, and CTCL, the infiltrating T cells could be stained with mAbs to alpha 3 or beta 1 and were localized in close proximity to the epiligrin-containing basement membrane. Infiltrating lymphocytes in malignant cutaneous B disease (CBCL) did not express alpha 3 beta 1 by immunohistochemical techniques and did not associate with the epidermal basement membrane. The present findings clearly define a function for alpha 3 beta 1 in T cells and strongly suggest that alpha 3 beta 1 interaction with epiligrin may be involved in the pathogenesis of cutaneous inflammation.

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Overexpression of hTLX3 in Association with Mutant IL7Rα Is Sufficient to Generate T-ALL In Vivo and to Transform Thymocytes in Vitro

Blood ◽

10.1182/blood-2020-140060 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 21-21

Author(s):

Gisele Olinto Libanio Rodrigues ◽

Julie Hixon ◽

Hila Winer ◽

Erica Matich ◽

Caroline Andrews ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

T Cell ◽

Cell Line ◽

Lymphoblastic Leukemia ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Cell Counts

Mutations of the IL-7Rα chain occur in approximately 10% of pediatric T-cell acute lymphoblastic leukemia cases. While we have shown that mutant IL7Ra is sufficient to transform an immortalized thymocyte cell line, mutation of IL7Ra alone was insufficient to cause transformation of primary T cells, suggesting that additional genetic lesions may be present contributing to initiate leukemia. Studies addressing the combinations of mutant IL7Ra plus TLX3 overexpression indicates in vitro growth advantage, suggesting this gene as potential collaborative candidate. Furthermore, patients with mutated IL7R were more likely to have TLX3 or HOXA subgroup leukemia. We sought to determine whether combination of mutant hIL7Ra plus TLX3 overexpression is sufficient to generate T-cell leukemia in vivo. Double negative thymocytes were isolated from C57BL/6J mice and transduced with retroviral vectors containing mutant hIL7R plus hTLX3, or the genes alone. The combination mutant hIL7R wild type and hTLX3 was also tested. Transduced thymocytes were cultured on the OP9-DL4 bone marrow stromal cell line for 5-13 days and accessed for expression of transduced constructs and then injected into sublethally irradiated Rag-/- mice. Mice were euthanized at onset of clinical signs, and cells were immunophenotyped by flow cytometry. Thymocytes transduced with muthIL-7R-hTLX3 transformed to cytokine-independent growth and expanded over 30 days in the absence of all cytokines. Mice injected with muthIL7R-hTLX3 cells, but not the controls (wthIL7R-hTLX3or mutIL7R alone) developed leukemia approximately 3 weeks post injection, characterized by GFP expressing T-cells in blood, spleen, liver, lymph nodes and bone marrow. Furthermore, leukemic mice had increased white blood cell counts and presented with splenomegaly. Phenotypic analysis revealed a higher CD4-CD8- T cell population in the blood, bone marrow, liver and spleen compared in the mutant hIL7R + hTLX3 mice compared with mice injected with mutant IL7R alone indicating that the resulting leukemia from the combination mutant hIL7R plus hTLX3 shows early arrest in T-cell development. Taken together, these data show that oncogenic IL7R activation is sufficient for cooperation with hTLX3 in ex vivo thymocyte cell transformation, and that cells expressing the combination muthIL7R-hTLX3 is sufficient to trigger T-cell leukemia in vivo. Figure Disclosures No relevant conflicts of interest to declare.

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Immune function of the blood-brain barrier: incomplete presentation of protein (auto-)antigens by rat brain microvascular endothelium in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.110.5.1757 ◽

1990 ◽

Vol 110 (5) ◽

pp. 1757-1766 ◽

Cited By ~ 100

Author(s):

W Risau ◽

B Engelhardt ◽

H Wekerle

Keyword(s):

T Cells ◽

Endothelial Cells ◽

T Cell ◽

T Lymphocytes ◽

Rat Brain ◽

Blood Brain Barrier ◽

T Cell Activation ◽

Cell Activation ◽

Ifn Gamma

The endothelial blood-brain barrier (BBB) has a critical role in controlling lymphocyte traffic into the central nervous system (CNS), both in physiological immunosurveillance, and in its pathological aberrations. The intercellular signals that possibly could induce lymphocytes to cross the BBB include immunogenic presentation of protein (auto-)antigens by BBB endothelia to circulating T lymphocytes. This concept has raised much, though controversial, attention. We approached this problem by analyzing in vitro immunospecific interactions between clonal rat T lymphocyte lines with syngeneic, stringently purified endothelial monolayer cultures from adult brain micro-vessels. The rat brain endothelia (RBE) were established from rat brain capillaries using double collagenase digestion, density gradient fractionation and selective cytolysis of contaminating pericytes by anti-Thy 1.1 antibodies and complement. Incubation with interferon-gamma in most of the brain-derived endothelial cells induced Ia-antigens in the cytoplasm and on the cell surface in some of the cells. Before the treatment, the cells were completely Ia-negative. Pericytes were unresponsive to IFN-gamma treatment. When confronted with syngeneic T cell lines specific for protein (auto-)antigens (e.g., ovalbumin and myelin basic protein, MBP), RBE were completely unable to induce antigen-specific proliferation of syngeneic T lymphocytes irrespective of pretreatment with IFN-gamma and of cell density. RBE were inert towards the T cells, and did not suppress T cell activation induced by other "professional" antigen presenting cells (APC) such as thymus-derived dendritic cells or macrophages. IFN-gamma-treated RBE were, however, susceptible to immunospecific T cell killing. They were lysed by MBP-specific T cells in the presence of the specific antigen or Con A. Antigen dependent lysis was restricted by the appropriate (MHC) class II product. We conclude that the interaction of brain endothelial cells with encephalitogenic T lymphocytes may involve recognition of antigen in the molecular context of relevant MHC products, but that this interaction per se is insufficient to initiate the full T cell activation program.

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