Linking Two Immuno-Suppressive Molecules: Indoleamine 2,3 Dioxygenase Can Modify HLA-G Cell-Surface Expression1

Nonclassical human leukocyte antigen (HLA) class I molecule HLA-G and indoleamine 2,3 dioxygenase (INDO) in humans and mice, respectively, have been shown to play crucial immunosuppressive roles in fetal-maternal tolerance. HLA-G inhibits natural killer and T cell function by high-affinity interaction with inhibitory receptors, and INDO acts by depleting the surrounding microenvironment of the essential amino acid tryptophan, thus inhibiting T cell proliferation. We investigated whether HLA-G expression and INDO function were linked. Working with antigen-presenting cell (APC) lines and monocytes, we found that functional inhibition of INDO by 1-methyl-tryptophan induced cell surface expression of HLA-G1 by HLA-G1- negative APCs that were originally cell-surface negative, and that in reverse, the functional boost of INDO by high concentrations of tryptophan induced a complete loss of HLA-G1 cell surface expression by APCs that were originally cell-surface HLA-G1-positive. This mechanism was shown to be posttranslational because HLA-G protein cell contents remained unaffected by the treatments used. Furthermore, HLA-G cell surface expression regulation by INDO seems to relate to INDO function, but not to tryptophan catabolism itself. Potentia

HLA-G gene editing in tumor cell lines as a novel alternative in cancer immunotherapy

Cancer immunotherapies based mainly on the blockade of immune-checkpoint (IC) molecules by anti-IC antibodies offer new alternatives for treatment in oncological diseases. However, a considerable proportion of patients remain unresponsive to them. Hence, the development of novel clinical immunotherapeutic approaches and/or targets are crucial.W In this context, targeting the immune-checkpoint HLA-G/ILT2/ILT4 has caused great interest since it is abnormally expressed in several malignancies generating a tolerogenic microenvironment. Here, we used CRISPR/Cas9 gene editing to block the HLA-G expression in two tumor cell lines expressing HLA-G, including a renal cell carcinoma (RCC7) and a choriocarcinoma (JEG-3). Different sgRNA/Cas9 plasmids targeting HLA-G exon 1 and 2 were transfected in both cell lines. Downregulation of HLA-G was reached to different degrees, including complete silencing. Most importantly, HLA-G − cells triggered a higher in vitro response of immune cells with respect to HLA-G + wild type cells. Altogether, we demonstrated for the first time the HLA-G downregulation through gene editing. We propose this approach as a first step to develop novel clinical immunotherapeutic approaches in cancer.Fil: Palma, Maria Belen. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Departamento de Ciencias Morfológicas. Cátedra de Citología y Embriología A; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; ArgentinaFil: Tronik Le Roux, Diana. Saint-Louis Hospital; Francia. Universite de Paris 1 - Pantheon Sorbonne.; FranciaFil: Amin, Guadalupe. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Castañeda, Sheila Lucia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Möbbs, Alan Miqueas. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Scarafia, Maria Agustina. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: la Greca, Alejandro Damián. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Daouya, Marina. Hôpital Saint-louis; Francia. Universite de Paris 1 - Pantheon Sorbonne.; FranciaFil: Poras, Isabelle. Hôpital Saint-louis; Francia. Universite de Paris 1 - Pantheon Sorbonne.; FranciaFil: Inda, Ana María. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Departamento de Ciencias Morfológicas. Cátedra de Citología y Embriología A; Argentina. Provincia de Buenos Aires. Gobernación. Comisión de Investigaciones Científicas; ArgentinaFil: Moro, Lucía Natalia. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Carosella, Edgardo Delfino. Hôpital Saint-louis; Francia. Universite de Paris 1 - Pantheon Sorbonne.; FranciaFil: García, Marcela Nilda. Universidad Nacional de La Plata. Facultad de Ciencias Médicas. Departamento de Ciencias Morfológicas. Cátedra de Citología y Embriología A; ArgentinaFil: Miriuka, Santiago Gabriel. Fundación para la Lucha contra las Enfermedades Neurológicas de la Infancia; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentin

Multimeric structures of HLA-G isoforms function through differential binding to LILRB receptors

The non-classical Human leukocyte antigen G (HLA-G) differs from classical HLA class I molecules by its low genetic diversity, a tissue-restricted expression, the existence of seven isoforms, and immuno-inhibitory functions. Most of the known functions of HLA-G concern the membrane-bound HLA-G1 and soluble HLA-G5 isoforms, which present the typical structure of classical HLA class I molecule: a heavy chain of three globular domains α(1)-α(2)-α(3) non-covalently bound to β-2-microglobulin (B2M) and a peptide. Very little is known of the structural features and functions of other HLA-G isoforms or structural conformations other than B2M-associated HLA-G1 and HLA-G5. In the present work, we studied the capability of all isoforms to form homomultimers, and investigated whether they could bind to, and function through, the known HLA-G receptors LILRB1 and LILRB2. We report that all HLA-G isoforms may form homodimers, demonstrating for the first time the existence of HLA-G4 dimers. We also report that the HLA-G α(1)-α(3) structure, which constitutes the extracellular part of HLA-G2 and HLA-G6, binds the LILRB2 receptor but not LILRB1. This is the first report of a receptor for a truncated HLA-G isoform. Following up on this finding, we show that the α(1)-α(3)-Fc structure coated on agarose beads is tolerogenic and capable of prolonging the survival of skin allografts in B6-mice and in a LILRB2-transgenic mouse model. This study is the first proof of concept that truncated HLA-G isoforms could be used as therapeutic agents

Tolerogenic Function of Dimeric Forms of HLA-G Recombinant Proteins: A Comparative Study In Vivo

HLA-G is a natural tolerogenic molecule involved in the best example of tolerance to foreign tissues there is: the maternal-fetal tolerance. The further involvement of HLA-G in the tolerance of allogeneic transplants has also been demonstrated and some of its mechanisms of action have been elucidated. For these reasons, therapeutic HLA-G molecules for tolerance induction in transplantation are actively investigated. In the present study, we studied the tolerogenic functions of three different HLA-G recombinant proteins: HLA-G heavy chain fused to β2-microglobulin (B2M), HLA-G heavy chain fused to B2M and to the Fc portion of an immunoglobulin, and HLA-G alpha-1 domain either fused to the Fc part of an immunoglobulin or as a synthetic peptide. Our results demonstrate the tolerogenic function of B2M-HLA-G fusion proteins, and especially of B2M-HLA-G5, which were capable of significantly delaying allogeneic skin graft rejection in a murine in vivo transplantation model. The results from our studies suggest that HLA-G recombinant proteins are relevant candidates for tolerance induction in human transplantation

Linking Two Immuno-Suppressive Molecules: Indoleamine 2,3 Dioxygenase Can Modify HLA-G Cell-Surface Expression 1

ABSTRAC

Primers used for fusion protein generation.

<p>Primers used for fusion protein generation.</p

Tolerogenic function of HLA-G fusion proteins and peptide.

<p><b>A.</b> C57BL/6 mice strongly recognize the MHC class II-disparate mutant bm12 mouse that carries the I-Abm12 alloantigen. The capability of HLA-G-coated beads to delay rejection was evaluated. Control treatment (dotted lines): beads coated with mAb but without HLA-G proteins. Results are expressed as Median of graft survival time. Kaplan-Meier curves representing graft survival are shown for each HLA-G protein/peptide for controls (plain lines). Associated values are indicated as a table underneath the curves. <b>B.</b> The same experiments were performed using ILT4-transgernic mice as skin graft recipients, and for Alpha1-Fc and B2M-HLA-G5. <b>Tables</b>: Median survival of transplant, number of animals, and significance are indicated below the corresponding survival graphs.</p