[Profile and immune environment of upper tract urothelial carcinoma]. / Profil et environnement immunitaires des tumeurs de la voie excrétrice supérieure.

INTRODUCTION AND OBJECTIVES: Upper Tract Urothelial Carcinoma (UTUC) are tumors that share similarities with bladder tumors. Immunotherapy is already used for bladder locations and appears to have interest for UTUC. In order to rationalize the immunotherapy development pipeline it seemed necessary to describe the immune infiltrate of a cohort of UTUC treated with nephroureterectomy and to determine the expression of a panel of immune checkpoints and co-stimulatory molecules on tumor cells as well as on infiltrating and circulating lymphocytes. MATERIALS AND METHODS: This is a monocentric, prospective and exploratory work. Patients treated with total nephroureterectomy or segmental ureterectomy for presumably infiltrative (≥ T1) UTUC managed at the Saint-Louis Hospital were included from January 2019 to July 2020. A set of markers and immune checkpoints were studied by flow fluorocytometry on circulating lymphocytes (PBMCs) and tumor-infiltrating lymphocytes (TILs). Some markers were also studied by immunohistochemistry on tumor sample. RESULTS: In total, 14 patients were included and 13 patients could be analyzed. 1 patient had no residual tumor. 5 tumors out of the 12 (41.7%) showed a lymphocytic inflammatory infiltrate. PD1 was the most represented checkpoint with a median expression rate of 41.4% on CD4+ TILs and 3.89% on circulating CD4+ T cells. This rate was 62.4% and 7.45% respectively on CD8+ T cells. TIGIT was the second most represented marker with a median expression rate on tumor-infiltrating CD4+ T cells of 25% and 2.9% on circulating CD4+ T cells. The median expression level of TIGIT on tumor-infiltrating CD8+ T cells was 23.3% and 3.2% on circulating CD8+ T cells. ICOS was highly expressed on CD4+ TILS with a median of 33.9% in contrast to CD8+ TILS (median: 6.67%). Variable expression of other checkpoints (ILT2, TIM3, LAG3 and OX40) was found without clear trend. CONCLUSION: This exploratory work highlighted that PD1 was the most represented checkpoint. TIGIT was the second most represented checkpoint while ICOS, TIM3 and LAG3 were 3 other checkpoints whose expression was found to be less important. ILT2 and OX40 appeared to be weakly expressed. LEVEL OF EVIDENCE: II.

Cytometry-based analysis of HLA-G functions according to ILT2 expression.

HLA-G was described as a molecule inhibiting NK and T cells functions through its receptor, ILT2. However, most functional studies of HLA-G were so far performed on heterogeneous immune populations and regardless of ILT2 expression. This may lead to an underestimation of the effect of HLA-G. Thus, considering the immune subpopulations sensitive to HLA-G remained an important issue in the field. Here we present a new cytometry assay to evaluate HLA-G effects on both NK and CD8+ T cell cytotoxic functions. Using flow cytometry allows for the comparison of HLA-G function on multiple subsets and multiple functions in the same time. In particular, we sharpen the analysis by specifically studying the immune subpopulations expressing HLA-G receptor ILT2. We focused our work on: IFN-gamma production and cytotoxicity (CD107a expression) by CD8+ T cells and NK cells expressing or not ILT2. We compared the expression of these markers in presence of target cells, expressing or not HLA-G1, and added a blocking antibody to reverse HLA-G inhibition. This new method allows for the discrimination of cell subsets responding and non-responding to HLA-G1 in one tube. We confirm that HLA-G-specifically inhibits the ILT2+ CD8+ T cell and ILT2+ NK cell subsets but not ILT2-negative ones. By blocking HLA-G/ILT2 interaction using an anti-ILT2 antibody we restored the cytotoxicity level, corroborating the specific inhibition of HLA-G1. We believe that our methodology enables to investigate HLA-G immune functions easily and finely towards other immune cell lineages or expressing other receptors, and might be applied in several pathological contexts, such as cancer and transplantation.

The role of methylation, DNA polymorphisms and microRNAs on HLA-G expression in human embryonic stem cells.

The human leukocyte antigen (HLA)-G gene seems to play a pivotal role in maternal tolerance to the fetus. Little is known about HLA-G expression and its molecular control during in vivo human embryogenesis. Human embryonic stem cells (hESC) provide an interesting in vitro model to study early human development. Different studies reported discrepant findings on whether HLA-G mRNA and protein are present or absent in hESC. Several lines of evidence indicate that promoter CpG methylation and 3' untranslated region (3'UTR) polymorphisms may influence HLA-G expression. We investigated how HLA-G expression is linked to the patterns of promoter methylation and explored the role of the 3'UTR polymorphic sites and their binding microRNAs on the post-transcriptional regulation of HLA-G in eight hESC lines. We showed that, while the gross expression levels of HLA-G are controlled by promoter methylation, the genetic constitution of the HLA-G 3'UTR, more specifically the 14bp insertion in combination with the +3187A/A and +3142G/G SNP, plays a major role in HLA-G mRNA regulation in hESC. Our findings provide a solid first step towards future work using hESC as tools for the study of early human developmental processes in normal and pregnancy-related disorders such as preeclampsia.

Recent advances on the non-classical major histocompatibility complex class I HLA-G molecule.

The human leukocyte antigen (HLA)-G non-classical major histocompatibility complex (MHC) class I molecule was originally described in first-trimester trophoblasts at the fetal-maternal interface in 1990. Eight years later, the First International Conference on this molecule was inaugurated by Prof Jean Dausset, recipient of the Nobel Prize in Medicine. The Fifth International Conference on HLA-G, held in Paris on July 2009, began with a tribute to Prof Jean Dausset who left us recently. This conference was co-chaired by Dr Edgardo D. Carosella and Prof Hans Grosse-Wilde, included 57 oral presentations and was attended by approximately 140 delegates from 16 countries. We summarize here the major advances on the HLA-G molecule that were reported, including findings on its biological activity and characterization of new mechanisms of action, notably through mesenchymal stem cells and regulatory cells, and the previously unexplored role of HLA-G on immune cells such as gammadelta T-cells and B lymphocytes. Furthermore, the role of HLA-G during pregnancy was revisited and its impact in pathologies such as cancer, autoimmune disorders and transplantation was further extended.

Research on HLA-G: an update.

Human leukocyte antigen (HLA)-G is a nonclassical HLA class I molecule from the major histocompatibility complex, which was initially shown to confer protection to the fetus from her mother's immune system. The Third International Conference on HLA-G, held in 2003, showed that beyond its role in fetal-maternal tolerance, HLA-G exerts tolerogenic functions involved in transplant acceptance as well as in tumoral and viral immune escape. The Fourth International Conference, which took place in Paris on July 2006, counted 72 oral presentations and about 200 attendees from 25 countries. The reports presented brought new insight into HLA-G research, and we summarize here the major advances on the HLA-G biology that were reported. Abstracts for all presentations can be found in volume 68 issue number 4 of Tissue Antigens.

Quantification and identification of soluble HLA-G isoforms.

In order to clarify the diagnostic relevance of soluble human leukocyte antigen-G (sHLA-G) molecules, reliable methods for the measurement of sHLA-G in various body fluids are of interest. Therefore, the aims of the 'Wet-Workshop for Quantification of Soluble HLA-G' held in Essen, Germany (at the Institute of Immunology, 18-20 October 2004) were to select and to validate HLA-G-specific enzyme-linked immunosorbent assay (ELISA) formats and purified standard HLA-G proteins, which can be easily generated and used as consensual references. We chose two ELISA formats, one for the simultaneous determination of shed HLA-G1 + sHLA-G5 (sHLA-G1 + G5) and one for the exclusive detection of HLA-G5 molecules. The first ELISA uses the antibody pair monoclonal antibody (mAb) MEM-G/9 + anti-beta2-microglobulin (beta2m), whereas the latter uses mAbs 5A6G7 + W6/32. Purified and well-defined HLA-G5 protein derived from insect SF9 cells transfected with HLA-G5 + human beta2m served as standard reagent. Twenty-five members of 13 international laboratories participated in the 3-day Wet-Workshop. The workshop demonstrated that the HLA-G5 protein was equally detected by both ELISA formats allowing direct comparison of quantitative results obtained by these two ELISA formats, and that sHLA-G1 + G5 and HLA-G5 molecules, respectively, were specifically and reproducibly quantified by the two ELISA formats. The comparison of the two ELISA results obtained allows the conclusion that sHLA-G1 and HLA-G5 molecules can exist in the blood of healthy donors. Moreover, there was evidence for a novel soluble HLA-G structure recognized by the mAbs 5A6G7 + W6/32 antibody combination but not by the one of mAb MEM-G/9 + anti-beta2m.

Functions of HLA-G in the immune system.

Human leukocyte antigen (HLA)-G is a nonclassical HLA class I molecule that has tolerogenic functions and acts on cells of both innate and adaptive immunity. The molecular mechanism leading to tolerance involves the interaction between HLA-G and inhibitory receptors that are expressed at the surface of immune cells. In this review, we will briefly summarize the key advances on the relationships between HLA-G and the immune system and their consequences in pathology.

Biology and functions of human leukocyte antigen-G in health and sickness.

In 1998, the first International Conference on human leukocyte antigen-G (HLA-G) was held in Paris. At that time, HLA-G was still a new HLA class I molecule, few aspects of its immunological functions were known, and its expression by tumors was just being described. In 1998, tools to properly study HLA-G were lacking, especially monoclonal antibodies, and three conclusions were drawn after the congress: (i) animal models were needed, (ii) the biology of HLA-G isoforms had to be confirmed, and (iii) HLA-G expression by tumors required clarification. Five years later, these three issues have been addressed. HLA-G is now gaining pace and is investigated for its immuno-inhibitory functions in the context of multiple pathologies. Eighty five oral presentations were given this year for more than 200 investigators working on HLA-G by speakers from over 20 countries. The success of the 3rd International Conference on HLA-G reflects the interest and tremendous work of the many research teams which, over the years, contributed to the publication of more than 500 peer-review articles. We summarize the key points that were presented and discussed during this meeting.

CD8+CD28- T suppressor cells and the induction of antigen-specific, antigen-presenting cell-mediated suppression of Th reactivity.

Human CD8+CD28- suppressor T cells (Ts) are a subset of T cells generated in the course of in vitro and in vivo immunizations. Ts recognize MHC class I:peptide complexes and inhibit the reactivity of T helper cells (Th) with cognate antigen specificity. We have demonstrated for the first time that CD8+CD28- Ts represent a unique subset of regulatory cells that induces the differentiation of tolerogenic antigen-presenting cells, initiating a suppressive loop which results in the induction and spreading of Th unresponsiveness.

Distinct mRNA microarray profiles of tolerogenic dendritic cells.

Dendritic cells are crucial to the activation as well as suppression of the immune response. Previous reports have illustrated that APC interacting with antigen-specific T suppressor cells become tolerogenic, inducing T helper anergy. To characterize the molecular changes occurring in tolerogenic APC, the mRNA profile of KG-1 dendritic cells exposed to allospecific T helper and T suppressor cells were analyzed. This study now provides evidence that immature dendritic cells stimulated by T suppressor cells differentiate into mature dendritic cells with a distinct phenotype. The identification of Ts induced pathways of dendritic cell differentiation is critical to the development of new therapeutic strategies.

Functional evidence that conserved TCR CDR alpha 3 loop docking governs the cross-recognition of closely related peptide:class I complexes.

The TCR recognizes its peptide:MHC (pMHC) ligand by assuming a diagonal orientation relative to the MHC helices, but it is unclear whether and to what degree individual TCRs exhibit docking variations when contacting similar pMHC complexes. We analyzed monospecific and cross-reactive recognition by diverse TCRs of an immunodominant HVH-1 glycoprotein B epitope (HSV-8p) bound to two closely related MHC class I molecules, H-2K(b) and H-2K(bm8). Previous studies indicated that the pMHC portion likely to vary in conformation between the two complexes resided at the N-terminal part of the complex, adjacent to peptide residues 2-4 and the neighboring MHC side chains. We found that CTL clones sharing TCR beta-chains exhibited disparate recognition patterns, whereas those with drastically different TCRbeta-chains but sharing identical TCRalpha CDR3 loops displayed identical functional specificity. This suggested that the CDRalpha3 loop determines the TCR specificity in our model, the conclusion supported by modeling of the TCR over the actual HSV-8:K(b) crystal structure. Importantly, these results indicate a remarkable conservation in CDRalpha3 positioning, and, therefore, in docking of diverse TCRalphabeta heterodimers onto variant peptide:class I complexes, implying a high degree of determinism in thymic selection and T cell activation.

Age-related dysregulation in CD8 T cell homeostasis: kinetics of a diversity loss.

Relative diversity and representation of peripheral T cells bearing different TCR Vbeta families are remarkably tightly regulated between birth and advanced adulthood. By contrast, individual elderly humans and C3H.SW and B10.BR aged mice display drastic disruption in such regulation. It was suggested that the alterations in the murine aged T cell compartment were due to age-related clonal T cell expansions (TCE). Here, we studied the kinetics of homeostatic dysregulation of T cell populations in aged C57BL/6 (B6) mice. Using mAb staining, we show that the percentages of alphabeta+CD8+ or CD4+ T cells bearing different TCRVbeta elements remain virtually constant in mice up to 12 mo of age. In 22-mo-old mice, however, there is a dramatic disturbance of this pattern owing to the emergence of CD8+ TCE. Expanded T cells did not show any obvious bias in Vbeta usage and were derived in all cases examined thus far from a single clone. TCE appeared later in life, compared with B cell clonal expansions. However, and in contrast to those detected in humans, TCE were frequently unstable disappearing within 2-4 mo, with other TCE appearing within the same time frame. Additional studies carried on thymic T cells, thymectomized mice, and young T transferred cells into Rag1-/- mice suggest that the clonal expansions occur in the periphery and that their onset is accelerated by decreased thymic output and/or function(s).

The mode of ligand recognition by two peptide:MHC class I-specific monoclonal antibodies.

The Ig superfamily members TCR and B cell receptor (BCR) share high structural and amino acid homology, yet interact with Ags in a distinct manner. The overall shape of the TCR ligand is rather constant, with the variation coming from the MHC polymorphism and the peptide heterogeneity. Consequently, the TCR alpha- and beta-chains form a relatively flat ligand-binding site that interacts with the peptide:MHC (pep:MHC) ligand in a fixed diagonal orientation relative to the MHC alpha-helices, with the alpha- and beta-chains of the TCR contacting the N and C termini of the pep:MHC complex, respectively. By contrast, the shape of BCR ligands varies dramatically, and the BCR exhibits much greater variability of the Ag-binding site. The mAbs 25-D1.16 (D1) and 22-C5.9 (C5), specific for the OVA-8:H-2Kb complex, allowed us to directly compare how TCR and BCR approach the same ligand. To that effect, we mapped D1 and C5 footprints over the OVA-8:H-2Kb complex. Using peptide variants and mutant MHC molecules, we show that the D1 and C5 contacts with the OVA-8:Kb complex C terminus overlap with the TCR beta-chain footprint, but that this footprint also extends to the regions of the molecule not contacted by the TCR. These studies suggest that D1 and C5 exhibit a hybrid mode of pep:MHC recognition, in part similar to that of the TCR beta-chain and in part similar to the conventional anti-MHC Ab.

Cellular basis of B cell clonal populations in old mice.

Previous studies from this laboratory have shown that >85% of old mice have stable B cell clonal populations detectable by Ig heavy chain complementary-determining region 3 mRNA size analysis and confirmed by sequence analysis. B cells from the same clone are frequently detected in several lymphoid compartments of the same mouse. We now report the phenotype of all ten stable B cell clonal populations detected in five 20-month-old C57BL/6 mice. These clonal B cells appear to develop in the periphery and nine of the ten B cell clonal populations expressed the CD5 cell surface marker. Stable B cell expansions may be dominated by cells at two stages of differentiation. Some B cell populations were detected with DNA as well as RNA and represent large clonal populations of B cells, detectable in several lymphoid compartments. These populations are found predominantly in B cell populations expressing CD45R/B220 and the mRNA coding for the membrane-bound form of the mu Ig heavy chain, which suggests a predominance of B lymphocytes in these populations. In other cases, smaller clonal populations were detected only in splenic RNA samples. These clonal populations were found predominantly among CD45R/B220- B cells and did not express the membrane-bound form of the micro Ig heavy chain. We offer the hypothesis that the B cell clonal populations present in old mice may be precursors of the two types of B cell neoplasms which are dominated by CD5+ B cells (B cell chronic lymphocytic leukemia) or plasma cells (multiple myeloma).

Heteroclitic immunization induces tumor immunity.

In tumor transplantation models in mice, cytotoxic T lymphocytes (CTLs) are typically the primary effector cells. CTLs recognize major histocompatibility complex (MHC) class I-associated peptides expressed by tumors, leading to tumor rejection. Peptides presented by cancer cells can originate from viral proteins, normal self-proteins regulated during differentiation, or altered proteins derived from genetic alterations. However, many tumor peptides recognized by CTLs are poor immunogens, unable to induce activation and differentiation of effector CTLs. We used MHC binding motifs and the knowledge of class I:peptide:TCR structure to design heteroclitic CTL vaccines that exploit the expression of poorly immunogenic tumor peptides. The in vivo potency of this approach was demonstrated using viral and self-(differentiation) antigens as models. First, a synthetic variant of a viral antigen was expressed as a tumor antigen, and heteroclitic immunization with peptides and DNA was used to protect against tumor challenge and elicit regression of 3-d tumors. Second, a peptide from a relevant self-antigen of the tyrosinase family expressed by melanoma cells was used to design a heteroclitic peptide vaccine that successfully induced tumor protection. These results establish the in vivo applicability of heteroclitic immunization against tumors, including immunity to poorly immunogenic self-proteins.

Increased VH 11 and VH Q52 gene use by splenic B cells in old mice associated with oligoclonal expansions of CD5 + B cells.

A significant increase in the utilization of the VH gene families VH11 and Q52 was observed in LPS-stimulated splenic B lymphocytes from aged mice compared to young mice. VH gene usage was assayed by in situ DNA/RNA hybridization using VH family-specific and kappa chain probes. The observed age-dependent differences appear to reflect the preferential use of VH11 and Q52 VH gene use by the CD5 + B lymphocyte subset whose numbers in the spleen increase with age. The increased use of VH11 by splenic cells from old mice is associated with clonal expansions of splenic CD5 + B lymphocytes.

Clonal expansions of B lymphocytes in old mice.

The effect of age on the diversity of the murine Ig heavy chain repertoire has been studied in unimmunized C57BL/6 mice. We examined the heterogeneity of complementarity-determining region 3 (CDR3) sizes of Ig mRNA of the IgM and IgG isotypes using two VH families, VHJ558 and VHQ52, which together account for approximately 65% of the Ab repertoire. The broad and bell-shaped profiles representing the diversity of the VHJ558 family in the spleen of 2- to 6-mo-old C57BL/6 mice becomes significantly less diverse after 12 mo of age and by 18 mo of age, single CDR3 sizes that dominate the profiles can be observed in the spleens of > 85% of the mice. Readable sequences have been obtained from 40 dominant mRNA CDR3 size species indicating that they represent clonal populations of B lineage. There are no significant homologies among these sequences. Clones of B lymphocytes that express a dominant CDR3 mRNA species can also be found in the bone marrow, the mesenteric lymph nodes, and the thymus of C57BL/6 mice > 18 mo of age. Some clones of B cells can be detected in only one lymphoid compartment; others are found in two or more compartments. The splenic B cell clones in C57BL/6 mice > 18 mo of age are stable for at least 2 mo. The CDR3 mRNA species that dominate the splenic repertoire of Ig mRNA-expressing cells in vivo do not dominate the repertoire of splenic B cells activated in vitro by bacterial LPS, suggesting that they represent a modest population of B cells expressing high levels of Ig mRNA.

Effect of age on humoral immunity, selection of the B-cell repertoire and B-cell development.

The age-associated changes in humoral immunity affect the quality more than the quantity of the antibody response. Changes in the quality of the antibody response with age include shifts in antibody specificities from foreign to autoantigens, in antibody isotypes from IgG to IgM, in antibody affinities from high to low and in the antibody idiotypic repertoire. These changes can be traced to an impaired capacity of T cells to facilitate: (a) the maturation of B cells with respect to isotype and affinity maturation in the periphery and (b) the development of a diverse B-cell repertoire from precursors within the bone marrow. In contrast, there is no evidence that the amount of immunoglobulin produced before or after immunization diminishes with age. Nonetheless, the impaired responses of the elderly to most vaccines and the greater susceptibility of the elderly to infections has fostered a view that immune senescence leads to a state of immune deficiency. However, it is more precise to describe immune senescence as leading to a state of immune dysregulation. The dysregulation of the humoral immunity is manifested by a shift from adaptive humoral immunity, characterized by the production of a highly specific, high-affinity, IgG antibody response to foreign antigens, to a process of natural antibody-mediated immunity, dominated by low-affinity, polyreactive, IgM antibodies which react with autoantigens. Age-associated T-cell impairments appear to be the basis for the shift from adaptive to natural humoral immunity and their reversal should permit the restoration of an adaptive antibody response in the elderly.