We describe a process for rapid antibody affinity optimization by repertoire mining to identify clones across B cell clonal lineages based on convergent immune responses where antigen-specific clones with the same heavy (VH) and light chain germline segment pairs, or parallel lineages, bind a single epitope on the antigen. We use this convergence framework to mine unique and distinct VH lineages from rat anti-triggering receptor on myeloid cells 2 (TREM2) antibody repertoire datasets with high diversity in the third complementarity-determining loop region (CDR H3) to further affinity-optimize a high-affinity agonistic anti-TREM2 antibody while retaining critical functional properties. Structural analyses confirm a nearly identical binding mode of anti-TREM2 variants with subtle but significant structural differences in the binding interface. Parallel lineage repertoire mining is uniquely tailored to rationally explore the large CDR H3 sequence space in antibody repertoires and can be easily and generally applied to antibodies discovered in vivo.
© 2024. The Author(s).

Cytomegaloviruses manipulate the host chemokine/receptor axis by altering cellular chemokine expression and by encoding multiple chemokines and chemokine receptors. Similar to human cytomegalovirus (HCMV), rat cytomegalovirus (RCMV) encodes multiple CC chemokine-analogous proteins, including r129 (HCMV UL128 homologue) and r131 (HCMV UL130 and MCMV m129/130 homologues). Although these proteins play a role in CMV entry, their function as chemotactic cytokines remains unknown. In the current study, we examined the role of the RCMV chemokine r129 in promoting cellular migration and in accelerating transplant vascular sclerosis (TVS) in our rat heart transplant model. We determined that r129 protein is released into culture supernatants of infected cells and is expressed with late viral gene kinetics during RCMV infection and highly expressed in heart and salivary glands during in vivo rat infections. Using the recombinant r129 protein, we demonstrated that r129 induces migration of lymphocytes isolated from rat peripheral blood, spleen, and bone marrow and from a rat macrophage cell line. Using antibody-mediated cell sorting of rat splenocytes, we demonstrated that r129 induces migration of naïve/central memory CD4(+) T cells. Through ligand-binding assays, we determined that r129 binds rat CC chemokine receptors CCR3, CCR4, CCR5, and CCR7. In addition, mutational analyses identified functional domains of r129 resulting in recombinant proteins that fail to induce migration (r129-ΔNT and -C31A) or alter the chemotactic ability of the chemokine (r129-F43A). Two of the mutant proteins (r129-C31A and -ΔNT) also act as dominant negatives by inhibiting migration induced by wild-type r129. Furthermore, infection of rat heart transplant recipients with RCMV containing the r129-ΔNT mutation prevented CMV-induced acceleration of TVS. Together our findings indicate that RCMV r129 is highly chemotactic, which has important implications during RCMV infection and reactivation and acceleration of TVS.

The objective of the present study was to examine the effects of dietary Zn deficiency on the ex vivo cytokine production (IL-2, interferon-gamma (IFN-gamma), IL-6 and IL-10) of isolated thymocytes and splenocytes after mitogenic stimulation with concavalin A and to explore the role of corticosterone in this regulation. Weanling rats were assigned to one of four dietary treatments for 3 weeks: Zn-deficient (< 1mg Zn/kg diet, ad libitum), pair-fed (30 mg Zn/kg diet, limited to amount of feed as consumed by the Zn-deficient group), marginally Zn-deficient (10 mg Zn/kg diet, ad libitum) and control (30 mg Zn/kg diet, ad libitum). Thymocytes and splenocytes were isolated for cytokine stimulation and determination of T-cell phenotypes. Serum corticosterone concentrations were determined by ELISA. The Zn-deficient and pair-fed groups had 14-fold higher serum corticosterone concentrations compared with the marginally Zn-deficient and control groups (P<0.0001). The proportions of thymocyte subsets were not altered in the Zn-deficient, pair-fed or marginally Zn-deficient groups; however, thymocyte IL-2 and IL-6 production in these groups was 33-54% lower compared with the control group (P<0.05). The Zn-deficient group had an 18-28% lower proportion of new T-cells (TCRalphabeta+CD90+), but no difference in the proportion of new T-cells that were cytotoxic or helper. The Zn-deficient group had a 49-62% lower production of Th1 cytokines (IL-2), but no difference in the production of Th2 cytokines (IL-6, IL-10) by stimulated splenocytes compared with the pair-fed, marginally Zn-deficient and control groups (P<0.01). These results indicate that Zn status is associated with altered cytokine production, while in vivo corticosterone concentrations are not associated with ex vivo cytokine production.