# Molecular characterization of the functional isoforms of HLA-F in human health and cancer

> **NIH NIH R21** · UNIVERSITY OF CHICAGO · 2020 · $237,048

## Abstract

Project Summary/Abstract
HLA-F is a nonclassical class I MHC (Ib) molecule that has been found expressed on a variety of cancers,
shown to play a role in HIV infection and the neurological autoimmune disease ALS, and is expressed
throughout pregnancy. Despite the potential importance of this protein in these conditions, little is known about
this molecule in terms of its function or even in which state it is expressed. We have recently shown that, in
addition to being expressed as a heavy chain only state, or open conformer (HLA-FOC), HLA-F can also be
expressed as a bona fide peptide presenting molecule, associated with the β2m subunit (pHLA-F). Peptides
are presented in an unconventional way, with the N-terminus not anchored within the groove and the potential
for post-translational modifications featuring in peptide anchoring. Despite these advances, there remains
much unknown about the role for presented peptide in HLA-F function and how HLA-F engages its various
receptors in each of these states. Thus, the aims of this proposal focus on addressing these questions and are:
Aim 1: Develop and validate nanobodies towards peptide-loaded (pHLA-F) and open conformer (HLA-
FOC) states of HLA-F and determine their expression profile in cancer cell lines. As HLA-F has been
described to function in both the peptide-bound and free states, we will develop and validate novel nanobodies
that specifically bind to these different forms of HLA-F. We will validate them through the use of HLA-F over
expressing and knock out cell lines by flow cytometry, immunoprecipitation, and western blot. Finally, validated
nanobody-binders will be used to determine the dominant form of HLA-F (HLA-FOC, pHLA-F, or both)
expressed in and on the surface of 1) standard cell lines and 2) from gynecologic, neuroblastoma, and
hepatocellular carcinoma cancer cell lines. Aim 2: Determine the peptide-antigen repertoire of HLA-F from
selected cancer cell lines and determine their effect on NK-cell receptor engagement. As we have
previously published, we have the ability to produce recombinant pHLA-F from HEK293T cells using an
engineered baculovirus. We also showed that we can use this purified protein to determine the peptide
repertoire of HLA-F from this cell line using MS/MS. Using this approach, we will produce pHLA-F from the
cancer cell lines mentioned in SA1 and determine the peptide repertoire from these cancer specific lineages. In
addition, we will pull out endogenous protein from these lines and confirm our results or act as an alternative
approach should recombinant protein prove difficult to produce. Finally, we will determine the role of these
peptide-antigens in NK-cell receptor engagement using in vitro binding experiments.

## Key facts

- **NIH application ID:** 9958682
- **Project number:** 1R21AI151956-01
- **Recipient organization:** UNIVERSITY OF CHICAGO
- **Principal Investigator:** Erin June Adams
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $237,048
- **Award type:** 1
- **Project period:** 2020-01-20 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9958682

## Citation

> US National Institutes of Health, RePORTER application 9958682, Molecular characterization of the functional isoforms of HLA-F in human health and cancer (1R21AI151956-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9958682. Licensed CC0.

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