OR.96. MHC Class I-Restricted Human Melanoma Epitope Specific TCR Engineered Human CD4 T Cells Exhibit Anti-Tumor Effector Function, Proliferate upon Cognate Stimulation, Amplify CD8+CTL Generation, and Mitigate Inducible T Regulatory Cell (iTreg) Activities in Human Melanoma, in vitro

Abstract Abstract 3152 Alloreactive immune responses directed against malignant cells in recipients of allogeneic hematopoietic stem cell transplants are able to cure patients with hematological cancers. However, such immune responses may cause severe morbidity when directed against healthy recipient tissue, resulting in graft-versus-host disease (GvHD). Naturally occurring regulatory T cells (Tregs) are CD4+ T cells characterized by their expression of the transcription factor Foxp3. Whilst adoptively transferred polyclonal Tregs suppress GvHD in several murine models, their lack of specificity may compromise beneficial immunity against malignancy or infection. The generation of MHC class I-restricted, alloantigen-specific Tregs would allow them to recognize antigen presented directly on GvHD target tissues, concentrating their sites of activation at these tissues and possibly reducing the potential for non-specific immune suppression. We have generated ‘converted’ Tregs through retroviral transfer of genes encoding Foxp3 and specific MHC class I-restricted T cell receptors (TCRs) into polyclonal conventional CD4+ T cells. We used the 2C-TCR, which recognizes the MHC class I molecule H-2Ld, expressed in Balb/c and other H-2d mice, in complex with the ubiquitously expressed peptide p2Ca; and the MH-TCR, which recognizes the MHC class I molecule H-2Db, expressed in B6 and other H-2b mice, in complex with the male peptide WMHHNMDLI. In vitro, Foxp3 2C-TCR-transduced B6 polyclonal CD4+ T cells were hyporesponsive to stimulation and suppressed the alloreactive proliferative response of B6 CD4+ and CD8+ T cells to Balb/c splenocytes, consistent with the acquisition of regulatory function. When adoptively transferred to lethally irradiated Balb/c recipients of MHC-mismatched B6 bone marrow and conventional T cells, Foxp3 2C-TCR-transduced B6 polyclonal CD4+ T cells significantly reduced early proliferation of donor T cells, weight loss and GvHD score in the recipients. Similarly, polyclonal CD4+ T cells transduced with Foxp3 and the MH-TCR caused marked suppression of allogeneic responses both in vitro and in vivo. However, while both the 2C-TCR and the MH-TCR conferred specificity to their cognate antigens in vitro, the potent suppression in these in vivo models was independent of the cognate antigen for the transduced TCRs. This non-specific suppression was markedly reduced when class I-restricted TCRs were transduced into OT-II Rag1-/- CD4+ T cells that are transgenic for a single endogenous TCR. These findings demonstrate an important role for the endogenous TCRs in driving non-specific suppression by polyclonal CD4+ T cells transduced with Foxp3 and class I-restricted TCRs, and suggest that strategies to downregulate endogenous TCRs will be required to achieve antigen-specific suppression in TCR gene-modified regulatory T cells. Disclosures: No relevant conflicts of interest to declare.

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Alloreactive CD8 T Cells Process and Present Acquired MHC Class I Alloantigen To Indirect Pathway Helper CD4 T Cells.

Transplantation Journal ◽

10.1097/00007890-201407151-00979 ◽

2014 ◽

Vol 98 ◽

pp. 310

Author(s):

E. Wlodek ◽

A. Jason ◽

K. Saeb-Parsy ◽

M. Chhubra ◽

G. Pettigrew

Keyword(s):

T Cells ◽

Mhc Class I ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Class I ◽

Indirect Pathway

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Immunotherapy of a murine tumor with interleukin 2. Increased sensitivity after MHC class I gene transfection.

Journal of Experimental Medicine ◽

10.1084/jem.166.6.1716 ◽

1987 ◽

Vol 166 (6) ◽

pp. 1716-1733 ◽

Cited By ~ 61

Author(s):

J S Weber ◽

G Jay ◽

K Tanaka ◽

S A Rosenberg

Keyword(s):

T Cells ◽

Mhc Class I ◽

Interleukin 2 ◽

Class I ◽

High Dose ◽

Class I Mhc ◽

High Doses ◽

I Gene

We have shown that two weakly immunogenic MCA sarcomas developed in our laboratory that are sensitive to high-dose IL-2 immunotherapy express class I MHC in vivo and in vitro. Two nonimmunogenic MCA sarcomas are relatively insensitive to IL-2 therapy and express minimal or no class I MHC molecules in vivo and in vitro. To study the role of MHC in the therapy of tumors with IL-2, a class I-deficient murine melanoma, B16BL6, was transfected with the Kb class I gene. Expression of class I MHC rendered B16BL6 advanced pulmonary macrometastases sensitive to IL-2 immunotherapy. 3-d micrometastases of CL8-2, a class I transfected clone of B16BL6, were significantly more sensitive to IL-2 therapy than a control nontransfected line. Expression of Iak, a class II MHC molecule, had no effect on IL-2 therapy of transfectant pulmonary micrometastases in F1 mice. By using lymphocyte subset depletion with mAbs directed against Lyt-2, therapy of class I transfectant macrometastases with high-dose IL-2 was shown to involve an Lyt-2 cell. In contrast, regression of micrometastases treated with low-dose IL-2 involved Lyt-2+ cells, but regression mediated by high doses of IL-2 did not. We hypothesize that both LAK and Lyt-2+ T cells effect IL-2-mediated elimination of micrometastases, but only Lyt-2+ T cells are involved in macrometastatic regression. Low doses of IL-2 stimulate Lyt-2+ cells to eliminate class I-expressing micrometastases, but high doses of IL-2 can recruit LAK cells to mediate regression of micrometastases independent of class I expression. Only high-dose IL-2, mediating its effect predominantly via Lyt-2+ cells, is capable of impacting on MHC class I-expressing macrometastases. Macrometastases devoid of class I MHC antigens appear to be resistant to IL-2 therapy.

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T Cell Receptor Gene Transfer to Produce MHC Class I-Restricted CD4 Helper T Cells: Implications for Immunotherapy of Leukaemia.

Blood ◽

10.1182/blood.v104.11.5262.5262 ◽

2004 ◽

Vol 104 (11) ◽

pp. 5262-5262

Author(s):

Emma Morris ◽

Aristotle Tsallios ◽

Gavin Bendle ◽

Shao-an Xue ◽

Hans Stauss

Keyword(s):

T Cells ◽

Mhc Class I ◽

Cd4 T Cells ◽

Tumour Cells ◽

Class I ◽

Helper T Cells ◽

Specific Response ◽

Cd4 Cells ◽

Cd8 Cells ◽

Irrelevant Peptide

Abstract CD4 helper T cells play a critical role in the anti-tumour immune response. Cytokines secreted by CD4 T cells can have a direct effect on tumour cells and provide help for CTL priming and effector function. In this study we tested if it was possible to generate MHC class I-restricted helper T cells by retroviral TCR gene transfer into CD4 lymphocytes. Methods: We used a TCR (utilising V11) that recognises the influenza virus A nucleoprotein (NP366–379) peptide in the context of murine Db MHC class I. Murine splenocytes were isolated from C57BL/6 mice (H2b) and activated with conconavalin A and IL-7, and after 48 hours transduced with the pMX-TCR-IRES-TCR retroviral vector. The transduced splenocytes were then cultured in the presence of IL2 for a further 48 hours before staining with anti-murine CD4, CD8 and V11 antibodies and sorting into CD4+ V11+ and CD8+ V11+ populations. Sorted cells were expanded for a further 48–72 hours prior to functional assays. Functional Assays: Purified TCR-transduced (TCR-Td) CD8+ cells and purified TCR-Td CD4+ cells were tested for IFN secretion in response to dendritic cells (DCs) pulsed with NP peptide, an irrelevant peptide (pMDM100) or no peptide. Further experiments examined IFN secretion in response to peptide-loaded tumour cells (EL4 murine lymphoma cells) or transfected tumour cells expressing NP endogenously. Secretion of IFN was measured by ELISA. Results: (1) Antigen-specific IFN secretion was observed by both CD8+ (100% purity) and CD4+ cells (99.93% purity) expressing the class I-restricted TCR when incubated with peptide-loaded DCs. When tested with no peptide or irrelevant peptide, no IFN secretion was observed. The CD8+ cells were more sensitive, recognizing lower concentrations of peptide (10pM) than CD4+ cells (100pM). With peptide-coated EL4 tumour cells as stimulator cells, CD8+ cells showed a peptide-specific response. In contrast, the TCR-Td CD4+ cells were only able to elicit a weak peptide-specific response. Similarly, TCR-Td CD8+ cells were able to recognise NP transfected EL4 tumour cells (EL4NP68), whereas the CD4+ cells were unable to. However, the addition of syngeneic DCs restored the CD4+ cell response to NP transfected EL4 tumour cells to one equivalent to that seen with the TCR-Td CD8+ populations (Table 1). Summary: We have demonstrated that it is feasible to generate MHC class I-restricted CD4+ helper T cells, that are specific for peptide epitopes presented in the context of MHC class I. The CD4+ T cells can recognise antigen-expressing tumour cells in the presence of professional APC, such as DCs. The mechanism by which APC restore tumour recognition may involve trans-costimulation or cross presentation. The data suggest that class I-restricted CD4+ T cells may be able to contribute to enhanced anti-tumour immunity. αββββγγγγγβ γIFN Secretion (ng/ml) After Stimulation with DCs or Tumour Cells T Cell (Responder Cell) Stimulator Cell/s No Peptide NP (100nM) pMDM100 (100nM) Abbreviations: ND not done; DC, EL4 and EL4NP68 as indicated in text. TCR-Td CD8+ DCs 0.1 163.2 0.7 TCR-Td CD8+ EL4 0.1 19.9 0.2 TCR-Td CD8+ EL4NP68 16.6 ND ND TCR-Td CD8+ EL4NP68 + DCs 31.2 ND ND TCR-Td CD4+ DCs 0.1 163.9 0.2 TCR-Td CD4+ EL4 0.1 0.8 0.0 TCR-Td CD4+ EL4NP68 0.2 ND ND TCR-Td CD4+ EL4NP68 + DCs 25.3 ND ND

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Non-Cognate Cytotoxic T Cells Can Be Retargeted Against CD20 Positive Tumour Cells Using [Fab' × MHC Class I/Peptide] Conjugates.

Blood ◽

10.1182/blood.v116.21.2838.2838 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2838-2838

Author(s):

Angela D Hamblin ◽

Ben CR King ◽

Ruth R French ◽

Claude H Chan ◽

Alison L Tutt ◽

...

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Mhc Class I ◽

Peripheral Blood ◽

Class I ◽

B Cell Depletion ◽

Cytokine Release ◽

Anti Cd20

Abstract Abstract 2838 To circumvent cytotoxic T lymphocyte (CTL) tolerance of tumour-associated antigens, the concept of redirecting CTLs against non-cognate targets has developed. One way of doing this is to use bispecific antibodies comprising anti-CD3 and anti-tumour antigen moieties. Unfortunately, this is frequently associated with unacceptable toxicity due to inflammatory cytokine release. As an alternative our approach has been to use a bivalent conjugate recognising a tumour antigen (through an antibody fragment) and a defined population of CTLs (specific for a single antigenic peptide e.g. viral epitope) through peptide presented in the context of recombinant MHC class I. We have produced a conjugate consisting of an anti-human CD20 Fab' fragment joined via a chemical crosslinker (succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate) to murine MHC class I/peptide (Kbα1-α3 domains/β2microglobulin presenting the ovalbumin-derived peptide SIINFEKL; expressed bacterially as a continuous polypeptide single chain trimer after Yu et al, J Immunol 2002). Size exclusion chromatography allowed purification of conjugates with [Fab':MHC class I/peptide] ratios of 1:1 and 2:1 (F2 and F3 respectively). In vitro both constructs were able to redirect the transgenic murine CTL line OT-1 (specific for KbSIINFEKL) to lyse human CD20+ tumour cells (lymphoblastoid Daudi cell line) at effector: target ratios of 10:1. This lysis could be blocked by the addition of 100 fold excess of either anti-CD20 F(ab')2 or the Kb/SIINFEKL-specific antibody 25D1. The constructs were also able to cause in vitro proliferation of naïve OT-1 cells (but not irrelevant CD8+ T cells) in the presence of human CD20+ cells in both thymidine incorporation and CFSE dilution assays. Using a human CD20 transgenic mouse model (Ahuja et al, J Immunol 2007) we have evaluated both constructs in vivo for their ability to redirect adoptively transferred OT-1 cells to deplete B cells from the peripheral blood. A single dose of 1 nmole F3 and 2 nmole F2 caused respectively up to 95% and 85% B cell depletion at day 7. The efficacy of lower doses suggested a dose: response relationship. As a marker of toxicity, we have measured cytokine levels at 2, 8 and 24 hours following a dose of 1 nmole F3 and compared them to those seen after administration of an [anti-CD3 × anti-CD20] bispecific F(ab')2 at a dose (0.5 nmole) which produced similar day 7 peripheral blood B cell depletion: phosphate-buffered saline was given as a negative control. Maximal cytokine release was seen at 2 hours with the levels of IL-4, IL-5, KC, IL-2 and IL-10 being lower after administration of the F3 than after the bispecific F(ab')2. However, interestingly, the F3 resulted in greater IL-12 release. Overall these data suggest that [Fab' × MHC class I/peptide] constructs have the potential to redirect non-cognate CTLs to deplete CD20+ malignant B cells from the peripheral blood and that this is associated with a lower level of cytokine release than a similarly efficacious dose of an anti-CD3-containing bispecific F(ab')2. Furthermore, the ability of [Fab' × MHC class I/peptide] constructs to cause proliferation of OT-1 cells in vitro suggests it may be possible to use a single molecule to both generate a secondary cytotoxic T cell response and subsequently to retarget it, increasing the viability of the approach if adopted in the clinic. Disclosures: No relevant conflicts of interest to declare.

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Sialoadhesin-Positive Host Macrophages Play an Essential Role in Graft-Versus-Leukemia Reactivity in Mice

Blood ◽

10.1182/blood.v93.12.4375.412k32_4375_4386 ◽

1999 ◽

Vol 93 (12) ◽

pp. 4375-4386 ◽

Cited By ~ 1

Author(s):

Susanne Müerköster ◽

Marian Rocha ◽

Paul R. Crocker ◽

Volker Schirrmacher ◽

Victor Umansky

Keyword(s):

T Cells ◽

T Cell ◽

Mhc Class I ◽

Mhc Class Ii ◽

Cd8 T Cells ◽

Class I ◽

Peptide Epitope ◽

Peptide Epitopes ◽

Graft Versus Leukemia

We recently established an effective immune T-cell–mediated graft-versus-leukemia (GVL) murine model system in which complete tumor remissions were achievable even in advanced metastasized cancer. We now describe that this T-cell–mediated therapy is dependent on host macrophages expressing the lymphocyte adhesion molecule sialoadhesin (Sn). Depletion of Kupffer cells in tumor-bearing mice during adoptive immunotherapy (ADI) or the treatment of these animals with anti-Sn monoclonal antibodies led to complete or partial inhibition of the immune T-cell–mediated therapeutic effect. Furthermore, Sn+ host macrophages in livers formed clusters during ADI with donor CD8 T cells. To test for a possible antigen presentation function of these macrophages, we used as an in vitro model the antigen β-galactosidase for which a dominant major histocompatibility complex (MHC) class I Ld-restricted peptide epitope is known to be recognized by specific CD8 cytotoxic T lymphocytes (CTL). We demonstrate that purified Sn+ macrophages can process exogenous β-galactosidase and stimulate MHC class I peptide-restricted CTL responses. Thus, Sn+ macrophages, which are significantly increased in the liver after ADI, may process tumor-derived proteins via the MHC class I pathway as well as via the MHC class II pathway, as shown previously, and present respective peptide epitopes to CD8 as well as to CD4 immune T cells, respectively. The synergistic interactions observed before between immune CD4 and CD8 T cells during ADI could thus occur in the observed clusters with Sn+ host macrophages.

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