Defining Clinical and Immunological Predictors of Poor Immune Responses to COVID-19 mRNA Vaccines in Patients with Primary Antibody Deficiency.

Immune responses to coronavirus disease 2019 (COVID-19) mRNA vaccines in primary antibody deficiencies (PADs) are largely unknown. We investigated antibody and CD4+ T-cell responses specific for SARS-CoV-2 spike protein (S) before and after vaccination and associations between vaccine response and patients' clinical and immunological characteristics in PADs. The PAD cohort consisted of common variable immune deficiency (CVID) and other PADs, not meeting the criteria for CVID diagnosis (oPADs). Anti-S IgG, IgA, and IgG subclasses 1 and 3 increased after vaccination and correlated with neutralization activity in HCs and patients with oPADs. However, 42% of CVID patients developed such responses after the 2nd dose. A similar pattern was also observed with S-specific CD4+ T-cells as determined by OX40 and 4-1BB expression. Patients with poor anti-S IgG response had significantly lower levels of baseline IgG, IgA, CD19+ B-cells, switched memory B-cells, naïve CD8+ T-cells, and a higher frequency of EM CD8+ T-cells and autoimmunity compared to patients with adequate anti-S IgG responses. Patients with oPADs can develop humoral and cellular immune responses to vaccines similar to HCs. However, a subset of CVID patients exhibit impairment in developing such responses, which can be predicted by the baseline immune profile and history of autoimmunity.

Carbonyl Posttranslational Modification Associated With Early-Onset Type 1 Diabetes Autoimmunity.

Inflammation and oxidative stress in pancreatic islets amplify the appearance of various posttranslational modifications to self-proteins. In this study, we identified a select group of carbonylated islet proteins arising before the onset of hyperglycemia in NOD mice. Of interest, we identified carbonyl modification of the prolyl-4-hydroxylase ß subunit (P4Hb) that is responsible for proinsulin folding and trafficking as an autoantigen in both human and murine type 1 diabetes. We found that carbonylated P4Hb is amplified in stressed islets coincident with decreased glucose-stimulated insulin secretion and altered proinsulin-to-insulin ratios. Autoantibodies against P4Hb were detected in prediabetic NOD mice and in early human type 1 diabetes prior to the onset of anti-insulin autoimmunity. Moreover, we identify autoreactive CD4+ T-cell responses toward carbonyl-P4Hb epitopes in the circulation of patients with type 1 diabetes. Our studies provide mechanistic insight into the pathways of proinsulin metabolism and in creating autoantigenic forms of insulin in type 1 diabetes.

Vaccine-induced ErbB (EGFR/HER2)-specific immunity in spontaneous canine cancer.

Epidermal Growth Factor Receptor (EGFR) is overexpressed on a number of human cancers, and often is indicative of a poor outcome. Treatment of EGFR/HER2 overexpressing cancers includes monoclonal antibody therapy (cetuximab/trastuzumab) either alone or in conjunction with other standard cancer therapies. While monoclonal antibody therapy has been proven to be efficacious in the treatment of EGFR/HER2 overexpressing tumors, drawbacks include the lack of long-lasting immunity and acquired resistance to monoclonal therapy. An alternative approach is to induce a polyclonal anti-EGFR/HER2 tumor antigen response by vaccine therapy. In this phase I/II open-label study, we examined anti-tumor immunity in companion dogs with spontaneous EGFR expressing tumors. Canine cancers represent an outbred population in which the initiation, progression of disease, mutations and growth factors closely resemble that of human cancers. Dogs with EGFR expressing tumors were immunized with a short peptide of the EGFR extracellular domain with sequence homology to HER2. Serial serum analyses demonstrated high titers of EGFR/HER2 binding antibodies with biological activity similar to that of cetuximab and trastuzumab. Canine antibodies bound both canine and human EGFR on tumor cell lines and tumor tissue. CD8 T cells and IgG deposition were evident in tumors from immunized dogs. The antibodies inhibited EGFR intracellular signaling and inhibited tumor growth in vitro. Additionally, we illustrate objective responses in reducing tumors at metastatic sites in host animals. The data support the approach of amplifying anti-tumor immunity that may be relevant in combination with other immune modifying therapies such as checkpoint inhibitors.

Epidermal growth factor receptor peptide vaccination induces cross-reactive immunity to human EGFR, HER2, and HER3.

Current treatments for tumors expressing epidermal growth factor receptor (EGFR) include anti-EGFR monoclonal antibodies, often used in conjunction with the standard chemotherapy, radiation therapy, or other EGFR inhibitors. While monoclonal antibody treatment is efficacious in many patients, drawbacks include its high cost of treatment and side effects associated with multiple drug infusions. As an alternative to monoclonal antibody treatments, we have focused on peptide-based vaccination to trigger natural anti-tumor antibodies. Here, we demonstrate that peptides based on a region of the EGFR extracellular domain IV break immune tolerance to EGFR and elicit anti-tumor immunity. Mice immunized with isoforms of EGFR peptide p580-598 generated anti-EGFR antibody and T-cell responses. Iso-aspartyl (iso-Asp)-modified EGFR p580 immune sera inhibit in vitro growth of EGFR overexpressing human A431 tumor cells, as well as promote antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies induced by Asp and iso-Asp p580 bound homologous regions of the EGFR family members HER2 and HER3. EGFR p580 immune sera also inhibited the growth of the human tumor cell line MDA-MB-453 that expresses HER2 but not EGFR. Asp and iso-Asp EGFR p580 induced antibodies were also able to inhibit the in vivo growth of EGFR-expressing tumors. These data demonstrate that EGFR peptides from a region of the EGFR extracellular domain IV promote anti-tumor immunity, tumor cell killing, and antibodies that are cross reactive with ErbB family members.

Autoantigens: novel forms and presentation to the immune system.

It is clear that lupus autoimmunity is marked by a variety of abnormalities, including those found at a macroscopic scale, cells and tissues, as well as more microenvironmental influences, originating at the individual cell surface through to the nucleus. The convergence of genetic, epigenetic, and perhaps environmental influences all lead to the overt clinical expression of disease, reflected by the presences of autoantibodies and tissue pathology. This review will address several specific areas that fall among the non-genetic factors that contribute to lupus autoimmunity and related syndromes. In particular, we will discuss the importance of understanding various protein post-translational modifications (PTMs), mechanisms that mediate the ability of "modified self" to trigger autoimmunity, and how these PTMs influence lupus diagnosis. Finally, we will discuss altered pathways of autoantigen presentation that may contribute to the perpetuation of chronic autoimmune disease.

Lupus autoimmunity altered by cellular methylation metabolism.

Modifications of both DNA and protein by methylation are key factors in normal T and B cell immune responses as well as in the development of autoimmune disease. For example, the failure to maintain the methylation status of CpG dinucleotides in DNA triggers T cell autoreactivity. Methylated proteins are known targets of autoimmunity, including the symmetrical dimethylarginine residues of SmD1 and SmD3 in SLE. Herein, we demonstrate that altering the metabolism of S-adenosylmethionine (SAM), the major methyl donor for transmethylation reactions, can suppress T cell immunity. A by-product of SAM metabolism, 5'-deoxy-5'-methylthioadenosine (MTA), and an indirect inhibitor of methyltransferases, inhibits T cell responses including T cell activation markers, Th1/Th2 cytokines and TCR-related signaling events. Moreover, treatment of the lupus-prone MRL/lpr mouse with MTA markedly ameliorates splenomegaly, lymphadenopathy, autoantibody titers as well as IgG deposition and cellular infiltration in the kidney. Incubation of cells with SAM, which increases intracellular MTA levels, inhibits both TCR-mediated T cell proliferation and BCR (anti-IgM)-triggered B cell proliferation in a dose-dependent manner. These studies define the central role of MTA and SAM in immune responses and provide a simple approach to altering lymphocyte transmethylation and T cell mediated autoimmune syndromes.

Antigen presentation and transfer between B cells and macrophages.

B cells play an active role in directing immunity against specific proteins in part because of their capacity to sequester antigen via B cell receptor (BCR). Our prior findings indicate that B cells can initiate an immune response in vivo to self proteins independent of other antigen-presenting cells (APC). However, these studies also demonstrated that both dendritic cells and macrophages are important in the ongoing immune response. The present work illustrates a mechanism by which antigen acquired by B cells through BCR is specifically transferred to other APC, in particular, macrophages. The transfer of antigen is dependent on the specificity of BCR and requires direct contact between the cells, but does not require MHC compatibility between the cells and is independent of the activation state of macrophages. Antigen transfer is functional, in that macrophages, which received B cell derived-antigen, can activate CD4 T cells. Overall, these results define a novel mechanism by which B cells can focus immunity toward a specific antigen and transfer the ability to activate CD4 T cells to other APC.

Altered immunogenicity of isoaspartate containing proteins.

The development of immune tolerance is dependent on the expression of self-peptides in the thymus and bone marrow during lymphocyte development. However, not all self-antigens are expressed in the thymus, particularly for proteins that become post-translationally modified during other biological processes in a cell. We have found that one such post-translational modification, the spontaneous conversion of an aspartic acid to isoaspartic acid (isoAsp), causes ignored self-antigens to become immunogenic. In order to determine the mechanism for this autoimmune response, pigeon cytochrome c peptide 88-104 (PCC p88-104) was synthesized with and without an isoaspartyl residue. Each form was digested with cathepsin D, an enzyme involved in antigen processing. The products of cathepsin digestion were dramatically different between the two forms of self-protein suggesting that cryptic self-peptides may be revealed to the immune system by natural modifications to self-proteins. This observation also held true if whole PCC protein contained isoaspartyl residues was digested with cathespsin D. Additionally, AND transgenic TCR T cells (recognizing PCC 88-104) proliferated to a greater extent in response to isoaspartyl PCC as compared to the normal form of PCC. These finding demonstrate the importance of post-translational modifications in shaping autoimmune responses in and the development of tolerance to self-proteins.

B cells drive early T cell autoimmunity in vivo prior to dendritic cell-mediated autoantigen presentation.

Both B cells and dendritic cells (DCs) have been implicated as autoantigen-presenting cells in the activation of self-reactive T cells. However, most self-proteins are ubiquitously and/or developmentally expressed, making it difficult to determine the source and the exposure of autoantigens to APCs in a controlled manner. In this study, we have used an Ig transgenic mouse model to examine the mechanisms by which B cells and other APCs acquire and present lupus autoantigens in vivo. Targeting a lupus autoantigen, the small nuclear ribonucleoprotein particle D protein, to the BCR activates autoreactive T cells in the periphery. Our in vivo studies demonstrate that autoantigen-specific B cells, when present in the repertoire, are the first subset of APCs to capture and present self-proteins for activating T cells. Thereafter, DCs acquire self-Ag and become effective APCs for stimulating the same subsets of autoreactive T cells. This mechanism provides one explanation of how early steps in autoimmunity can focus responses, via BCR, at a small group of self-proteins among the total milieu of intracellular self-proteins. Subsequently, DCs and other professional APCs may then amplify and perpetuate the autoimmune T cell response.

Intracellular protein modification associated with altered T cell functions in autoimmunity.

Posttranslational protein modifications influence a number of immunologic responses ranging from intracellular signaling to protein processing and presentation. One such modification, termed isoaspartyl (isoAsp), is the spontaneous nonenzymatic modification of aspartic acid residues occurring at physiologic pH and temperature. In this study, we have examined the intracellular levels of isoAsp residues in self-proteins from MRL(+/+), MRL/lpr, and NZB/W F(1) mouse strains compared with nonautoimmune B10.BR mice. In contrast to control B10.BR or NZB/W mice, the isoAsp content in MRL autoimmune mice increased and accumulated with age in erythrocytes, brain, kidney, and T lymphocytes. Moreover, T cells that hyperproliferate to antigenic stimulation in MRL mice also have elevated intracellular isoAsp protein content. Protein l-isoaspartate O-methyltransferase activity, a repair enzyme for isoAsp residues in vivo, remains stable with age in all strains of mice. These studies demonstrate a role for the accumulation of intracellular isoAsp proteins associated with T cell proliferative defects of MRL autoimmune mice.

Isoaspartyl post-translational modification triggers anti-tumor T and B lymphocyte immunity.

A hallmark of the immune system is the ability to ignore self-antigens. In attempts to bypass normal immune tolerance, a post-translational protein modification was introduced into self-antigens to break T and B cell tolerance. We demonstrate that immune tolerance is bypassed by immunization with a post-translationally modified melanoma antigen. In particular, the conversion of an aspartic acid to an isoaspartic acid within the melanoma antigen tyrosinase-related protein (TRP)-2 peptide-(181-188) makes the otherwise immunologically ignored TRP-2 antigen immunogenic. Tetramer analysis of iso-Asp TRP-2 peptide-immunized mice demonstrated that CD8+ T cells not only recognized the isoaspartyl TRP-2 peptide but also the native TRP-2 peptide. These CD8+ T cells functioned as cytotoxic T lymphocytes, as they effectively lysed TRP-2 peptide-pulsed targets both in vitro and in vivo. Potentially, post-translational protein modification can be utilized to trigger strong immune responses to either tumor proteins or potentially weakly immunogenic pathogens.

A failure to repair self-proteins leads to T cell hyperproliferation and autoantibody production.

It is clear that many factors can perturb T cell homeostasis that is critical in the maintenance of immune tolerance. Defects in the molecules that regulate homeostasis can lead to autoimmune pathology. This simple immunologic concept is complicated by the fact that many self-proteins undergo spontaneous posttranslational modifications that affect their biological functions. This is the case in the spontaneous conversion of aspartyl residues to isoaspartyl residues, a modification occurring at physiological pH and under conditions of cell stress and aging. We have examined the effect of isoaspartyl modifications on the effector functions of T lymphocytes in vivo using mice lacking the isoaspartyl repair enzyme protein carboxyl methyltransferase (PCMT). PCMT(-/-) CD4(+) T cells exhibit increased proliferation in response to mitogen and Ag receptor stimulation as compared with wild-type CD4(+) T cells. Hyperproliferation is marked by increased phosphorylation of members of both the TCR and CD28 signaling pathways. Wild-type mice reconstituted with PCMT(-/-) bone marrow develop high titers of anti-DNA autoantibodies and kidney pathology typical of that found in systemic lupus erythematosus. These observations, coupled with the fact that humans have polymorphisms in the pcmt gene, suggest that isoaspartyl self-proteins may alter the maintenance of peripheral immune tolerance.