Proper development of long-lived memory CD4 T cells requires HLA-DO function.

Introduction: HLA-DO (DO) is an accessory protein that binds DM for trafficking to MIIC and has peptide editing functions. DO is mainly expressed in thymic medulla and B cells. Using biochemical experiments, our lab has discovered that DO has differential effects on editing peptides of different sequences: DO increases binding of DM-resistant peptides and reduces the binding of DM-sensitive peptides to the HLA-DR1 molecules. In a separate line of work, we have established that appropriate densities of antigen presentation by B cells during the contraction phase of an infection, induces quiescence in antigen experienced CD4 T cells, as they differentiate into memory T cells. This quiescence phenotype helps memory CD4 T cell survival and promotes effective memory responses to secondary Ag challenge. Methods: Based on our mechanistic understanding of DO function, it would be expected that if the immunodominant epitope of antigen is DM-resistant, presentation of decreased densities of pMHCII by B cells would lead to faulty development of memory CD4 T cells in the absence of DO. We explored the effects of DO on development of memory CD4 T cells and B cells utilizing two model antigens, H5N1-Flu Ag bearing DM-resistant, and OVA protein, which has a DM-sensitive immunodominant epitope and four mouse strains including two DO-deficient Tg mice. Using Tetramers and multiple antibodies against markers of memory CD4 T cells and B cells, we tracked memory development. Results: We found that immunized DR1+DO-KO mice had fewer CD4 memory T cells and memory B cells as compared to the DR1+DO-WT counterpart and had compromised recall responses. Conversely, OVA specific memory responses elicited in HA immunized DR1+DO-KO mice were normal. Conclusion: These results demonstrate that in the absence of DO, the presentation of cognate foreign antigens in the DO-KO mice is altered and can impact the proper development of memory cells. These findings provide new insights on vaccination design leading to better immune memory responses.

H2-O deficiency promotes regulatory T cell differentiation and CD4 T cell hyperactivity.

Regulatory T cells (Treg) are crucial immune modulators, yet the exact mechanism of thymic Treg development remains controversial. Here, we present the first direct evidence for H2-O, an MHC class II peptide editing molecular chaperon, on selection of thymic Tregs. We provide evidence that lack of H2-O in the thymic medulla promotes thymic Treg development and leads to an increased peripheral Treg frequency. Single-cell RNA-sequencing (scRNA-seq) analysis of splenic CD4 T cells revealed not only of an enrichment of effector-like Tregs but also of activated CD4 T cells in the absence of H2-O. Our data support two concepts; a) lack of H2-O expression in the thymic medulla creates an environment permissive to Treg development and, b) that loss of H2-O drives increased basal auto-stimulation of CD4 T cells. These findings can help in better understanding of predispositions to autoimmunity and design of therapeutics for treatment of autoimmune diseases.

A cell-free antigen processing system informs HIV-1 epitope selection and vaccine design.

Distinct CD4+ T cell epitopes have been associated with spontaneous control of HIV-1 replication, but analysis of antigen-dependent factors that influence epitope selection is lacking. To examine these factors, we used a cell-free antigen processing system that incorporates soluble HLA-DR (DR1), HLA-DM (DM), cathepsins, and full-length protein antigens for epitope identification by LC-MS/MS. HIV-1 Gag, Pol, Env, Vif, Tat, Rev, and Nef were examined using this system. We identified 35 novel epitopes, including glycopeptides. Epitopes from smaller HIV-1 proteins mapped to regions of low protein stability and higher solvent accessibility. HIV-1 antigens associated with limited CD4+ T cell responses were processed efficiently, while some protective epitopes were inefficiently processed. 55% of epitopes obtained from cell-free processing induced memory CD4+ T cell responses in HIV-1+ donors, including eight of 19 novel epitopes tested. Thus, an in vitro processing system utilizing the components of Class II processing reveals factors influencing epitope selection of HIV-1 and represents an approach to understanding epitope selection from non-HIV-1 antigens.

H2-O deficiency promotes regulatory T cell differentiation and CD4 T cell hyperactivity.

Regulatory T cells (Treg) are crucial immune modulators, yet the exact mechanism of thymic Treg development remains controversial. Here, we present the first direct evidence for H2-O, an MHC class II peptide editing molecular chaperon, on selection of thymic Tregs. We identified that lack of H2-O in the thymic medulla promotes thymic Treg development and leads to an increased peripheral Treg frequency. Single-cell RNA-sequencing (scRNA-seq) analysis of splenic CD4 T cells revealed not only an enrichment of effector-like Tregs, but also activated CD4 T cells in the absence of H2-O. Our data support two concepts; a) lack of H2-O expression in the thymic medulla creates an environment permissive to Treg development and, b) that loss of H2-O drives increased basal auto-stimulation of CD4 T cells. These findings can help in better understanding of predispositions to autoimmunity and design of therapeutics for treatment of autoimmune diseases.

How Does B Cell Antigen Presentation Affect Memory CD4 T Cell Differentiation and Longevity?

Dendritic cells are the antigen presenting cells that process antigens effectively and prime the immune system, a characteristic that have gained them the spotlights in recent years. B cell antigen presentation, although less prominent, deserves equal attention. B cells select antigen experienced CD4 T cells to become memory and initiate an orchestrated genetic program that maintains memory CD4 T cells for life of the individual. Over years of research, we have demonstrated that low levels of antigens captured by B cells during the resolution of an infection render antigen experienced CD4 T cells into a quiescent/resting state. Our studies suggest that in the absence of antigen, the resting state associated with low-energy utilization and proliferation can help memory CD4 T cells to survive nearly throughout the lifetime of mice. In this review we would discuss the primary findings from our lab as well as others that highlight our understanding of B cell antigen presentation and the contributions of the MHC Class II accessory molecules to this outcome. We propose that the quiescence induced by the low levels of antigen presentation might be a mechanism necessary to regulate long-term survival of CD4 memory T cells and to prevent cross-reactivity to autoantigens, hence autoimmunity.

Multiple genetic programs contribute to CD4 T cell memory differentiation and longevity by maintaining T cell quiescence.

While memory T-cells represent a hallmark of adaptive immunity, little is known about the genetic mechanisms regulating the longevity of memory CD4 T cells. Here, we studied the dynamics of gene expression in antigen specific CD4 T cells during infection, memory differentiation, and long-term survival up to nearly a year in mice. We observed that differentiation into long lived memory cells is associated with increased expression of genes inhibiting cell proliferation and apoptosis as well as genes promoting DNA repair response, lipid metabolism, and insulin resistance. We identified several transmembrane proteins in long-lived murine memory CD4 T cells, which co-localized exclusively within the responding antigen-specific memory CD4 T cells in human. The unique gene signatures of long-lived memory CD4 T cells, along with the new markers that we have defined, will enable a deeper understanding of memory CD4 T cell biology and allow for designing novel vaccines and therapeutics.

The love and hate relationship of HLA-DM/DO in the selection of immunodominant epitopes.

Successful activation of CD4 T cells is centered around the ability of antigen presenting cells to successfully process, select Class II immunodominant epitopes from exogenous antigens and to present it to cognate T cells. To achieve this, newly synthesized MHC-II molecules are transferred to a specialized compartment which contain both exogenous antigens and the Class II processing machinery. Here in a process known as 'editing,' the Class II accessory molecule DM (HLA-DM human; murine H2-M) facilitates the loading and selection of exogenous peptides to MHC class II molecules thereby assuring proper selection of immunodominant epitopes. A second Class II accessory molecule, DO (HLA-DO human; murine H2-O), mainly present in B cells and thymic epithelium also contributes to the selection of immunodominant epitopes. Yet, despite a wealth of mechanistic insights into how DM functions, understanding the contributions of DO to epitope selection has proven to be highly challenging. In this review, we have attempted to discuss published in vitro and in vivo data during the past three years with insights into the biology of DO.

Lack of the MHC class II chaperone H2-O causes susceptibility to autoimmune diseases.

DO (HLA-DO, in human; murine H2-O) is a highly conserved nonclassical major histocompatibility complex class II (MHC II) accessory molecule mainly expressed in the thymic medulla and B cells. Previous reports have suggested possible links between DO and autoimmunity, Hepatitis C (HCV) infection, and cancer, but the mechanism of how DO contributes to these diseases remains unclear. Here, using a combination of various in vivo approaches, including peptide elution, mixed lymphocyte reaction, T-cell receptor (TCR) deep sequencing, tetramer-guided naïve CD4 T-cell precursor enumeration, and whole-body imaging, we report that DO affects the repertoire of presented self-peptides by B cells and thymic epithelium. DO induces differential effects on epitope presentation and thymic selection, thereby altering CD4 T-cell precursor frequencies. Our findings were validated in two autoimmune disease models by demonstrating that lack of DO increases autoreactivity and susceptibility to autoimmune disease development.