Interleukin-2 augmented activation of tumor associated macrophage plays the main role in MHC class I in vivo induction in tumor cells that are MHC negative in vitro

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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Augmentation of antitumor effects by NK cell inhibitory receptor blockade in vitro and in vivo

Blood ◽

10.1182/blood.v97.10.3132 ◽

2001 ◽

Vol 97 (10) ◽

pp. 3132-3137 ◽

Cited By ~ 106

Author(s):

Crystal Y. Koh ◽

Bruce R. Blazar ◽

Thaddeus George ◽

Lisbeth A. Welniak ◽

Christian M. Capitini ◽

...

Keyword(s):

Antitumor Activity ◽

Nk Cells ◽

Tumor Cells ◽

Mhc Class I ◽

Nk Cell ◽

Class I ◽

Inhibitory Receptors ◽

Antitumor Effects

Abstract Subsets of natural killer (NK) cells are characterized by the expression of inhibitory and/or stimulatory receptors specific for major histocompatibility complex (MHC) class I determinants. In mice, these include the Ly49 family of molecules. One mechanism by which tumor cells may evade NK cell killing is by expressing the appropriate MHC class I and binding inhibitory Ly49 receptors. Therefore, the question of whether blocking the interaction between the Ly49 inhibitory receptors on NK and MHC class I cells on tumor cells augments antitumor activity was investigated. Blockade of Ly49C and I inhibitory receptors using F(ab′)2 fragments of the 5E6 monoclonal antibody (mAb) resulted in increased cytotoxicity against syngeneic tumors and decreased tumor cell growth in vitro. The effect of 5E6 F(ab′)2 was specific for the MHC of the tumor, as the use of F(ab′)2 of the mAb against Ly49G2 failed to increase NK activity. Treatment of leukemia-bearing mice with 5E6 F(ab′)2 fragments or adoptive transfer of NK cells treated ex vivo with the F(ab′)2 resulted in significant increases in survival. These results demonstrate that blockade of NK inhibitory receptors enhances antitumor activity both in vitro and in vivo, suggesting that NK inhibitory receptors can be responsible for diminishing antitumor responses. Therefore, strategies to block inhibitory receptors may be of potential use in increasing the efficacy of immunotherapy.

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225 Optimal-affinity MAGE-A1-specific T cell receptors (TCRs) generated using the humanized TCR-transgenic mouse platform HuTCR are superior to human donor-derived TCRs

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.225 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A239-A239

Author(s):

Ioannis Gavvovidis ◽

Matthias Leisegang ◽

Vivian Scheuplein ◽

Matthias Obenaus ◽

Thomas Blankenstein ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Cells ◽

Mhc Class I ◽

Class I ◽

Human Donor ◽

High Affinity ◽

Cancer Testis

BackgroundAs cancer-testis antigens are self-antigens, T cells expressing high-affinity TCRs against such antigens are eliminated via negative selection. Therefore, human-derived TCRs are typically of low affinity and exhibit reduced anti-tumor activity. Affinity maturation by mutagenesis is a common tool to increase affinity but may result in reduced specificity and off-target toxicity. Using our proprietary HuTCR mouse platform, which consists of mouse lines carrying the full human TCR-a/ß loci and human HLA alleles, we have isolated naturally optimized high-affinity TCRs specific for the cancer-testis antigen MAGE-A1 and compared them in vitro and in vivo to human-derived MAGE-A1-specific TCRs that are currently reported to be in clinical development.MethodsMAGE-A1-specific TCRs were isolated from HuTCR mice immunized with the MAGE-1 antigen using scRNAseq or were synthesized based on publicly available databases of human donor-derived MAGE-A1-specific TCRs. All TCRs were re-expressed in primary human T cells as verified using peptide-MHC-multimer staining. Functional activity of the TCRs was analyzed by coculture with T2 target cells loaded with titrated amounts of epitope and measuring cytokine concentration by ELISA. Reactivity of TCRs to endogenously processed MAGE-A1 protein was assessed by coculture with tumor cell lines with variable MAGE-A1 and/or MHC-class-I expression. Tumor rejection potential of TCRs was evaluated in vivo using a syngeneic mouse model (TNA2 mice) expressing MAGE-A1 and HLA-A*02 on syngeneic tumor cells.ResultsImmunization of HuTCR mice with the MAGE-A1 antigen resulted in robust CD8+ T cell responses and several TCR clonotypes were identified by scRNAseq, with the majority of clonotypes being specific to the MAGE-A1-derived peptide KVLEYVIKV and TCR functional avidities ranging from 0.3nM to 3nM. In sharp contrast, human-derived TCRs of the same epitope specificity exhibited lower functional avidity with EC50 from 3nM to 60nM. In addition, HuTCR-mouse-derived TCRs were more sensitive in recognition of tumor cells expressing low MAGE-A1 and/or MHC-class-I. Adoptive T-cell transfer to TNA2-mice with established tumors resulted in complete rejection without relapse of tumors only in mice treated with HuTCR-mouse-derived TCR but not with human-derived or control TCRs.ConclusionsThe HuTCR mouse platform allows for the generation of high-affinity MAGE-A1-specific human TCRs with increased anti-tumor efficacy as compared to human-derived TCRs against the same cancer antigen. The in vitro and in vivo comparative data presented herein highlight the HuTCR-derived MAGE-A1-specific TCR as the most favorable candidate for clinical translation and a clinical trial evaluating its safety and efficacy in a variety of solid malignancies will be initiated November 2021.Ethics ApprovalAll animal experiments were performed according to institutional and national guidelines, after approval by the responsible authority (Landesamt für Gesundheit und Soziales, Berlin). Blood collection from healthy human donors was done after prior informed consent and experiments were conducted in accordance with the ethical standards of Declaration of Helsinki.

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Cross-priming of minor histocompatibility antigen-specific cytotoxic T cells upon immunization with the heat shock protein gp96.

Journal of Experimental Medicine ◽

10.1084/jem.182.3.885 ◽

1995 ◽

Vol 182 (3) ◽

pp. 885-889 ◽

Cited By ~ 249

Author(s):

D Arnold ◽

S Faath ◽

H Rammensee ◽

H Schild

Keyword(s):

Heat Shock ◽

Heat Shock Proteins ◽

Heat Shock Protein ◽

Tumor Cells ◽

Mhc Class I ◽

Donor Cell ◽

Class I ◽

Heat Shock Protein Gp96 ◽

Shock Proteins

Vaccination of mice with heat shock proteins isolated from tumor cells induces immunity to subsequent challenge with those tumor cells the heat shock protein was isolated from but not with other tumor cells (Udono, H., and P.K. Srivastava. 1994. J. Immunol. 152:5398-5403). The specificity of this immune response is caused by tumor-derived peptides bound to the heat shock proteins (Udono., H., and P.K. Srivastava. 1993. J. Exp. Med. 178:1391-1396). Our experiments show that a single immunization with the heat shock protein gp96 isolated from beta-galactosidase (beta-gal) expressing P815 cells (of DBA/2 origin) induces cytotoxic T lymphocytes (CTLs) specific for beta-gal, in addition to minor H antigens expressed by these cells. CTLs can be induced in mice that are major histocompatibility complex (MHC) identical to the gp96 donor cells (H-2d) as well as in mice with a different MHC (H-2b). Thus gp96 is able to induce "cross priming" (Matzinger, P., and M.J. Bevan. 1977. Cell. Immunol. 33:92-100), indicating that gp96-associated peptides are not limited to the MHC class I ligands of the gp96 donor cell. Our data confirm the notion that samples of all cellular antigens presentable by MHC class I molecules are represented by peptides associated with gp96 molecules of that cell, even if the fitting MHC molecule is not expressed. In addition, we extend previous reports on the in vivo immunogenicity of peptides associated gp96 molecules to two new groups of antigens, minor H antigens, and proteins expressed in the cytosol.

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Central Role of the Antigen-Presentation and Interferon-γ Pathways in Resistance to Immune Checkpoint Blockade

Annual Review of Cancer Biology ◽

10.1146/annurev-cancerbio-070220-111016 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Annette Paschen ◽

Ignacio Melero ◽

Antoni Ribas

Keyword(s):

Antigen Presentation ◽

Tumor Cells ◽

Mhc Class I ◽

Cancer Biology ◽

Interleukin 2 ◽

Interferon Γ ◽

Class I ◽

Annual Review ◽

Publication Date ◽

Type I

Resistance to immunotherapy is due in some instances to the acquired stealth mechanisms of tumor cells that lose expression of MHC class I antigen–presenting molecules or downregulate their class I antigen–presentation pathways. Most dramatically, biallelic β2-microglobulin (B2M) loss leads to complete loss of MHC class I expression and to invisibility to CD8+ T cells. MHC class I expression and antigen presentation are potently upregulated by interferon-γ (IFNγ) in a manner that depends on IFNγ receptor (IFNGR) signaling via JAK1 and JAK2. Mutations in these molecules lead to IFNγ unresponsiveness and mediate loss of recognition and killing by cytotoxic T lymphocytes. Loss of MHC class I augments sensitivity of tumor cells to be killed by natural killer (NK) lymphocytes, and this mechanism could be exploited to revert resistance, for instance, with interleukin-2 (IL-2)-based agents. Moreover, in some experimental models, potent local type I interferon responses, such as those following intratumoral injection of Toll-like receptor 9 (TLR9) or TLR3 agonists, revert resistance due to mutations of JAKs. Expected final online publication date for the Annual Review of Cancer Biology, Volume 6 is April 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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An adenoviral gene transfer system to express high levels of soluble allogeneic MHC class I molecules in vitro and in vivo

Transplantation Proceedings ◽

10.1016/s0041-1345(02)03252-9 ◽

2002 ◽

Vol 34 (6) ◽

pp. 2318-2319

Author(s):

C Graeb ◽

M Justl ◽

S Tange ◽

K.-W Jauch ◽

E.K Geissler

Keyword(s):

Gene Transfer ◽

Mhc Class I ◽

Class I ◽

Transfer System ◽

Adenoviral Gene Transfer ◽

Gene Transfer System ◽

Mhc Class I Molecules

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Soluble donor MHC class I antigen inhibits immunologic priming in vitro and in vivo

Transplant International ◽

10.1007/s001470050497 ◽

1998 ◽

Vol 11 (7) ◽

pp. S357-S360 ◽

Cited By ~ 2

Author(s):

E. K. Geissler ◽

M. N. Scherer ◽

C. Graeb

Keyword(s):

Mhc Class I ◽

Class I ◽

Mhc Class I Antigen

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Potential Immunocompetence of Proteolytic Fragments Produced by Proteasomes before Evolution of the Vertebrate Immune System

Journal of Experimental Medicine ◽

10.1084/jem.186.2.209 ◽

1997 ◽

Vol 186 (2) ◽

pp. 209-220 ◽

Cited By ~ 64

Author(s):

Gabriele Niedermann ◽

Rudolf Grimm ◽

Elke Geier ◽

Martina Maurer ◽

Claudio Realini ◽

...

Keyword(s):

Immune System ◽

Mhc Class I ◽

Structural Features ◽

Interferon Γ ◽

Class I ◽

Direct Products ◽

Vertebrate Immune System ◽

Mhc Class I Molecules

To generate peptides for presentation by major histocompatibility complex (MHC) class I molecules to T lymphocytes, the immune system of vertebrates has recruited the proteasomes, phylogenetically ancient multicatalytic high molecular weight endoproteases. We have previously shown that many of the proteolytic fragments generated by vertebrate proteasomes have structural features in common with peptides eluted from MHC class I molecules, suggesting that many MHC class I ligands are direct products of proteasomal proteolysis. Here, we report that the processing of polypeptides by proteasomes is conserved in evolution, not only among vertebrate species, but including invertebrate eukaryotes such as insects and yeast. Unexpectedly, we found that several high copy ligands of MHC class I molecules, in particular, self-ligands, are major products in digests of source polypeptides by invertebrate proteasomes. Moreover, many major dual cleavage peptides produced by invertebrate proteasomes have the length and the NH2 and COOH termini preferred by MHC class I. Thus, the ability of proteasomes to generate potentially immunocompetent peptides evolved well before the vertebrate immune system. We demonstrate with polypeptide substrates that interferon γ induction in vivo or addition of recombinant proteasome activator 28α in vitro alters proteasomal proteolysis in such a way that the generation of peptides with the structural features of MHC class I ligands is optimized. However, these changes are quantitative and do not confer qualitatively novel characteristics to proteasomal proteolysis. The data suggest that proteasomes may have influenced the evolution of MHC class I molecules.

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The Immunoevasive Function Encoded by the Mouse Cytomegalovirus Gene m152 Protects the Virus against T Cell Control in Vivo

Journal of Experimental Medicine ◽

10.1084/jem.190.9.1285 ◽

1999 ◽

Vol 190 (9) ◽

pp. 1285-1296 ◽

Cited By ~ 100

Author(s):

Astrid Krmpotic ◽

Martin Messerle ◽

Irena Crnkovic-Mertens ◽

Bojan Polic ◽

Stipan Jonjic ◽

...

Keyword(s):

Mhc Class I ◽

Gene Function ◽

Class I ◽

Viral Gene ◽

Mouse Cytomegalovirus ◽

Mcmv Infection ◽

Golgi Compartment ◽

Intermediate Compartment

Cytomegaloviruses encode numerous functions that inhibit antigen presentation in the major histocompatibility complex (MHC) class I pathway in vitro. One example is the mouse cytomegalovirus (MCMV) glycoprotein gp40, encoded by the m152 gene, which selectively retains murine but not human MHC class I complexes in the endoplasmic reticulum–Golgi intermediate compartment/cis-Golgi compartment (Ziegler, H., R. Thäle, P. Lucin, W. Muranyi, T. Flohr, H. Hengel, H. Farrell, W. Rawlinson, and U.H. Koszinowski. 1997. Immunity. 6:57–66). To investigate the in vivo significance of this gene function during MCMV infection of the natural host, we constructed recombinants of MCMV in which the m152 gene was deleted, as were the corresponding virus revertants. We report on the following findings: Deletion of the m152 gene has no effect on virus replication in cell culture, whereas after infection of mice, the m152-deficient virus replicates to significantly lower virus titers. This attenuating effect is lifted by reinsertion of the gene into the mutant. Mutants and revertants grow to the same titer in animals deprived of the function targeted by the viral gene function, namely in mice deficient in β2-microglobulin, mice deficient in the CD8 molecule, and mice depleted of T cells. Upon adoptive transfer of naive lymphocytes into infected mice, the absence of the m152 gene function sensitizes the virus to primary lymphocyte control. These results prove that MHC-reactive functions protect CMVs against attack by CD8+ T lymphocytes in vivo.

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