Targeting apoptosis to induce stable mixed hematopoietic chimerism and long-term allograft survival without myelosuppressive conditioning in mice.

Induction of mixed hematopoietic chimerism results in donor-specific immunological tolerance by apoptosis-mediated deletion of donor-reactive lymphocytes. A broad clinical application of this approach is currently hampered by limited predictability and toxicity of the available conditioning protocols. We developed a new therapeutic approach to induce mixed chimerism and tolerance by a direct pharmacological modulation of the intrinsic apoptosis pathway in peripheral T cells. The proapoptotic small-molecule Bcl-2 inhibitor ABT-737 promoted mixed chimerism induction and reversed the antitolerogenic effect of calcineurin inhibitors by boosting the critical role of the proapoptotic Bcl-2 factor Bim. A short conditioning protocol with ABT-737 in combination with costimulation blockade and low-dose cyclosporine A resulted in a complete deletion of peripheral donor-reactive lymphocytes and was sufficient to induce mixed chimerism and robust systemic tolerance across full major histocompatibility complex barriers, without myelosuppression and by using moderate doses of bone marrow cells. Thus, immunological tolerance can be achieved by direct modulation of the intrinsic apoptosis pathway in peripheral lymphocytes-a new approach to translate immunological tolerance into clinically applicable protocols.

Allelic variation in class I HLA determines CD8+ T cell repertoire shape and cross-reactive memory responses to SARS-CoV-2.

Effective presentation of antigens by human leukocyte antigen (HLA) class I molecules to CD8+ T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multiomic technology to generate a unified ex vivo characterization of the CD8+ T cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) across four major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCRα/ß sequence diversity, and the utilization of pre-existing SARS-CoV-2-reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations notably influence the CD8+ T cell repertoire shape and utilization of immune recall upon SARS-CoV-2 infection.

The BH3-mimetic ABT-737 inhibits allogeneic immune responses.

Apoptosis controls the adaptive immune system through regulation of central and peripheral lymphocyte deletion. Therefore, substances that selectively interact with the intrinsic apoptosis pathway in lymphocytes offer unexplored opportunities to pharmacologically modulate the immune response. Here, we present evidence that the BH3-mimetic ABT-737 suppresses allogeneic immune responses. In vitro, ABT-737 prevented allogeneic T-cell activation, proliferation, and cytotoxicity by apoptosis induction, but without impairing the physiological functions of remaining viable T cells. In vivo, ABT-737 was highly selective for lymphoid cells and inhibited allogeneic T- and B-cell responses after skin transplantation. The immunosuppressive effect of ABT-737 was markedly increased in combination with low-dose cyclosporine A, as shown by the induction of long-term skin graft survival without significant inflammatory infiltrates in 50% of the recipients in an MHC class I single antigen mismatched model. Thus, pharmacological targeting of Bcl-2 proteins represents a novel immunosuppressive approach to prevent rejection of solid organ allografts.

The Bcl-2 family antagonist ABT-737 significantly inhibits multiple animal models of autoimmunity.

The Bcl-2 family of proteins plays a critical role in controlling immune responses by regulating the expansion and contraction of activated lymphocyte clones by apoptosis. ABT-737, which was originally developed for oncology, is a potent inhibitor of Bcl-2, Bcl-x(L), and Bcl-w protein function. There is evidence that Bcl-2-associated dysregulation of lymphocyte apoptosis may contribute to the pathogenesis of autoimmunity and lead to the development of autoimmune diseases. In this study, we report that ABT-737 treatment resulted in potent inhibition of lymphocyte proliferation as measured by in vitro mitogenic or ex vivo Ag-specific stimulation. More importantly, ABT-737 significantly reduced disease severity in tissue-specific and systemic animal models of autoimmunity. Bcl-2 family antagonism by ABT-737 was efficacious in treating animal models of arthritis and lupus. Our results suggest that treatment with a Bcl-2 family antagonist represents a novel and potentially attractive therapeutic approach for the clinical treatment of autoimmunity.

Msh2 ATPase activity is essential for somatic hypermutation at a-T basepairs and for efficient class switch recombination.

Somatic hypermutation (SHM) and class switch recombination (CSR) are initiated by activation-induced cytidine deaminase-mediated cytidine deamination of immunoglobulin genes. MutS homologue (Msh) 2-/- mice have reduced A-T mutations and CSR. This suggests that Msh2 may play a role in repairing activation-induced cytidine deaminase-generated G-U mismatches. However, because Msh2 not only initiates mismatch repair but also has other functions, such as signaling for apoptosis, it is not known which activity of Msh2 is responsible for the effects observed, and consequently, many models have been proposed. To further dissect the role of Msh2 in SHM and CSR, mice with a "knockin" mutation in the Msh2 gene that inactivates the adenosine triphosphatase domain were examined. This mutation (i.e., Msh2G674A), which does not affect apoptosis signaling, allows mismatches to be recognized but prevents Msh2 from initiating mismatch repair. Here, we show that, similar to Msh2-/- mice, SHM in Msh2G674A mice is biased toward G-C mutations. However, CSR is partially reduced, and switch junctions are more similar to those of postmeiotic segregation 2-/- mice than to Msh2-/- mice. These results indicate that Msh2 adenosine triphosphatase activity is required for A-T mutations, and suggest that Msh2 has more than one role in CSR.

Examination of Msh6- and Msh3-deficient mice in class switching reveals overlapping and distinct roles of MutS homologues in antibody diversification.

Somatic hypermutation and class switch recombination (CSR) contribute to the somatic diversification of antibodies. It has been shown that MutS homologue (Msh)6 (in conjunction with Msh2) but not Msh3 is involved in generating A/T base substitutions in somatic hypermutation. However, their roles in CSR have not yet been reported. Here we show that Msh6(-)(/)(-) mice have a decrease in CSR, whereas Msh3(-)(/)(-) mice do not. When switch regions were analyzed for mutations, deficiency in Msh6 was associated with an increase in transition mutations at G/C basepairs, mutations at RGYW/WRCY hotspots, and a small increase in the targeting of G/C bases. In addition, Msh6(-)(/)(-) mice exhibited an increase in the targeting of recombination sites to GAGCT/GGGGT consensus repeats and hotspots in Sgamma3 but not in Smicro. In contrast to Msh2(-)(/)(-) mice, deficiency in Msh6 surprisingly did not change the characteristics of Smicro-Sgamma3 switch junctions. However, Msh6(-)(/)(-) mice exhibited a change in the positioning of Smicro and Sgamma3 junctions. Although none of these changes were seen in Msh3(-)(/)(-) mice, they had a higher percentage of large inserts in their switch junctions. Together, our data suggest that MutS homologues Msh2, Msh3, and Msh6 play overlapping and distinct roles during antibody diversification processes.

High antigen levels do not preclude B-cell tolerance induction to alpha1,3-Gal via mixed chimerism.

BACKGROUND: Studies of bone marrow transplantation (BMT) from wild-type mice or rats to alpha1,3-galactosyltransferase (GalT) knockout mice have demonstrated that induction of mixed chimerism tolerizes not only T cells, but also natural antibody-producing B cells, even across xenogeneic barriers. Given that rodent cells express lower levels of the alphaGal epitope than the more clinically relevant porcine species, the consequences of exposure to cells expressing high levels of alphaGal on the ability to induce B-cell tolerance are unknown. METHODS: The effects on chimerism and anti-alphaGal B-cell tolerance of an i.p. injection of 10(9) porcine RBC were evaluated in GalT knockout mice receiving wild-type allogeneic BMT after non-myeloablative conditioning with T-cell depleting monoclonal antibodies, thymic irradiation, and low-dose total body irradiation. RESULTS: Achievement of mixed chimerism and tolerance of anti-alphaGal-producing B cells was not affected by exposure to high-density alphaGal at the time of BMT. The absence of induced anti-alphaGal or anti-pig antibody responses in conditioned control mice suggested that the B-cell xeno-response to pig is T-cell-dependent. CONCLUSION: High alphaGal density on pig cells might not preclude the ability to achieve tolerance of pre-existing alphaGal-reactive human B cells via induction of mixed chimerism. This strategy has the potential to induce B-cell tolerance to non-alphaGal epitopes, against which natural antibodies have been found in the sera of healthy humans.

Potent and conditional redirected T cell killing of tumor cells using Half DVD-Ig.

Novel biologics that redirect cytotoxic T lymphocytes (CTLs) to kill tumor cells bearing a tumor associated antigen hold great promise in the clinic. However, the ability to safely and potently target CD3 on CTL toward tumor associated antigens (TAA) expressed on tumor cells remains a challenge of both technology and biology. Herein we describe the use of a Half DVD-Ig format that can redirect CTL to kill tumor cells. Notably, Half DVD-Ig molecules that are monovalent for each specificity demonstrated reduced non-specific CTL activation and conditional CTL activation upon binding to TAA compared to intact tetravalent DVD-Ig molecules that are bivalent for each specificity, while maintaining good drug like properties and appropriate PK properties.

Mutation detection of immunoglobulin V-regions by DHPLC.

One of the key features in the affinity maturation of antibodies is somatic hypermutation of the variable regions of immunoglobulin genes. The mutations that occur in immunoglobulin genes are detected by direct sequencing of cloned polymerase chain reaction (PCR) products. The frequencies of mutations in vivo are generally high enough to provide sufficient numbers of point mutations in order to generate large databases that can be analyzed in various ways. Recently, the mechanisms of variable (V)-region hypermutation have been studied in tissue culture systems and transgenic mice where mutation occurs at frequencies that are approximately 10-fold lower than the estimated in vivo rate. Identifying mutations by brute force sequencing of PCR products in comparative studies is limiting when trying to determine if there are statistically significant differences. Here we describe a high throughput technique that can facilitate the identification of immunoglobulin V-regions that contain one or more mutations before sequencing. This technique, known as denaturing high-performance liquid chromatography (DHPLC), utilizes a standard HPLC apparatus with a column that binds double-stranded DNA (dsDNA). In this study, we have successfully detected approximately 90% of previously sequenced mutated V-regions by DHPLC. Our results show that we were able to detect mutations throughout a 321-base pair (bp) region of the Ricin 45 immunoglobulin (Ig) V-region. Also, with the use of this assay, we have been able to detect mutations in multiple clones of different immunoglobulin genes.

B-cell extrinsic CR1/CR2 promotes natural antibody production and tolerance induction of anti-alphaGAL-producing B-1 cells.

B-1b cells produce IgM natural antibodies against alpha1-3Galbeta1-4GlcNAc (alphaGal). These can be tolerized by nonmyeloablative induction of mixed chimerism using alphaGal-positive (alphaGal+) donor marrow. We assessed the role of CR1/2 in this model for induction of tolerance of B-1b cells. Mixed hematopoietic chimerism was induced in alpha1-3galactosyltransferase (GalT-/-) and GalT-/-Cr2-/- mice with alphaGal+ BALB/c marrow donors. Anti-alphaGal Ab and anti-alphaGal Ab-producing B cells became undetectable in GalT-/- chimeras, whereas they persisted in chimeric GalT-/-Cr2-/- mice. To determine whether CR1/2 expression on stromal cells and/or hematopoietic cells was critical for B-1-cell tolerance, we generated GalT-/- radiation chimeras in which CR1/CR2 was expressed on either stromal cells, hematopoietic cells, neither, or both. After induction of mixed chimerism from alphaGal+ allogeneic bone marrow (BM) donors, anti-alphaGal-producing B cells were rendered tolerant in reconstituted recipients expressing only stromal CR1/CR2. Our results suggest a possible role for follicular dendritic cells that pick up immune complexes via CR1/CR2 receptors in the tolerization of B-1b cells.

Cutting edge: the G-U mismatch glycosylase methyl-CpG binding domain 4 is dispensable for somatic hypermutation and class switch recombination.

Affinity maturation of the humoral response is accomplished by somatic hypermutation and class switch recombination (CSR) of Ig genes. Activation-induced cytidine deaminase likely initiates these processes by deamination of cytidines in the V and switch regions of Ig genes. This activity is expected to produce G-U mismatches that can be substrates for MutS homolog 2/MutS homolog 6 heterodimers and for uracil DNA glycosylase. However, G-T and G-U mismatches are also substrates of the methyl-CpG binding domain 4 (Mbd4) glycosylase. To determine whether Mbd4 functions downstream of activation-induced cytidine deaminase activity, we examined somatic hypermutation and CSR in Mbd4(-/-) mice. In this study, we report that CSR, as analyzed by an in vitro switch assay and by in vivo immunizations, is unaffected in Mbd4(-/-) mice. In addition, the hypermutated JH2 to JH4 region in Peyer's patch B cells showed no effects as a result of Mbd4 deficiency. These data indicate that the Mbd4 glycosylase does not significantly contribute to mechanisms of Ab diversification.

Activation-induced cytidine deaminase turns on somatic hypermutation in hybridomas.

The production of high-affinity protective antibodies requires somatic hypermutation (SHM) of the antibody variable (V)-region genes. SHM is characterized by a high frequency of point mutations that occur only during the centroblast stage of B-cell differentiation. Activation-induced cytidine deaminase (AID), which is expressed specifically in germinal-centre centroblasts, is required for this process, but its exact role is unknown. Here we show that AID is required for SHM in the centroblast-like Ramos cells, and that expression of AID is sufficient to induce SHM in hybridoma cells, which represent a later stage of B-cell differentiation that does not normally undergo SHM. In one hybridoma, mutations were exclusively in G*C base pairs that were mostly within RGYW or WRCY motifs, suggesting that AID has primary responsibility for mutations at these nucleotides. The activation of SHM in hybridomas indicates that AID does not require other centroblast-specific cofactors to induce SHM, suggesting either that it functions alone or that the factors it requires are expressed at other stages of B-cell differentiation.

Altered somatic hypermutation and reduced class-switch recombination in exonuclease 1-mutant mice.

The generation of protective antibodies requires somatic hypermutation (SHM) and class-switch recombination (CSR) of immunoglobulin genes. Here we show that mice mutant for exonuclease 1 (Exo1), which participates in DNA mismatch repair (MMR), have decreased CSR and changes in the characteristics of SHM similar to those previously observed in mice mutant for the MMR protein Msh2. Exo1 is thus the first exonuclease shown to be involved in SHM and CSR. The phenotype of Exo1(-/-) mice and the finding that Exo1 and Mlh1 are physically associated with mutating variable regions support the idea that Exo1 and MMR participate directly in SHM and CSR.