Iranian Journal of Immunology

Abstract Immune checkpoint inhibitors have revolutionized cancer therapy by delivering long-lasting responses in a subset of patients across many cancer types. Yet, their effectiveness is often limited by high rates of primary and acquired resistance. This resistance is driven by complex interactions among tumor-intrinsic alterations, immunosuppressive factors within the tumor microenvironment, and host-related determinants. This review critically examines the biological mechanisms underlying resistance to immune checkpoint inhibitors, including defects in antigen presentation, dysregulated interferon signaling, activation of oncogenic pathways, compensatory upregulation of alternative immune checkpoints, and microbiome-associated immune modulation. In addition to defining these challenges, this review also highlights emerging opportunities for overcoming these obstacles. Emerging opportunities to circumvent resistance include biomarker-guided patient stratification, rational combination therapies that engage complementary immune pathways, modulation of the tumor microenvironment, and integration of multi-omics approaches to identify predictive resistance signatures. The central conclusion of this review is that effective clinical translation will necessitate a paradigm shift from discrete pathway inhibition toward integrated precision immuno-oncology strategies that combine molecular profiling, immune-contexture analysis, and mechanism-based combination therapies. Such integrated approaches may improve patient selection, overcome resistance, and expand the proportion of patients who achieve durable responses to immune checkpoint blockade.

Abstract Background: Pregnancy is a unique immunological state in which the semi-allogeneic fetus must be tolerated by the maternal immune system. Progesterone-induced blocking factor (PIBF) contributes to sustaining this immune balance through its regulatory effects, particularly those involving CD25⁺ T cells and maternal immune tolerance mechanisms.
Objective: To investigate whether PIBF influences the proportion of Foxp3+ cells among activated human peripheral CD4+ CD25+ T cells. 
Materials and methods: We isolated peripheral blood mononuclear cells (PBMCs) were isolated from 15 healthy women of reproductive-age, all sampled during the follicular phase of their menstrual cycles. The cells were stimulated with anti-CD3, anti-CD28 and IL-2 and cultured for 5 days in the presence or absence of PIBF at concentrations of 200 or 1000 ng/mL. The proportion of CD4+CD25+Foxp3+T cells was quantified by flow cytometry.
Results: Treatment with PIBF increased the percentage of CD4+CD25+Foxp3+ T cells compared with untreated controls (mean ± SE: control 2.7 ± 1.18%; PIBF 200 ng/mL 3.05 ± 1.06%, p = 0.008; PIBF 1000 ng/mL 3.50 ± 1.39%, p = 0.002). Although the increases were significant relative to the control group, no clear dose‑dependent trend was observed between the two PIBF concentrations. No adverse effects on cell viability were detected under the experimental conditions.
Conclusion: In vitro treatment of activated human peripheral CD4+CD25+ T cells with PIBF increased the percentage of Foxp3+ cells, suggesting that PIBF may promote regulatory T cell phenotypes in the context of maternal–fetal tolerance.

Abstract Background: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of myeloid cells that play a prominent role in maintaining immune tolerance. Despite considerable advances in reproductive immunology, current therapeutic regimens have failed provide a definitive solution for affected women, underscoring the imperative for continued investigation into the underlying mechanisms and novel intervention strategies.
Objective: To evaluate the effects of low-dose prednisolone therapy and lymphocyte immunotherapy (LIT) on MDSC subsets in women with recurrent miscarriage (RM) before pregnancy.
Methods: 100 women with RM were divided into two groups: a short-term low-dose prednisolone therapy group (n=50), and an LIT group (n=50). Blood samples were taken before and after the interventions and analyzed using flow cytometry. Specifically, we identified three MDSC subsets based on their surface markers: CD33+CD66+, CD15+CD66+, and CD14+CD66+ cells.
Results: LIT treatment significantly reduced CD33+CD66+ MDSCs (P=0.036). There were no significant differences in MDSC subsets before intervention, while prednisolone therapy increased granulocytic MDSCs (GR-MDSCs) compared to monocytic MDSCs (MO-MDSCs) in women with RM (P=0.046). In the LIT group, the decrease in the percentage of GR-MDSCs following treatment resulted in a significantly lower percentage of these cells compared to MO-MDSCs (P=0.0002).
Conclusion: Prednisolone increases the GR-MDSC to MO-MDSC ratio, while LIT decreases GR-MDSCs, shifting the balance toward MO-MDSCs. Both approaches have immunomodulatory effects in the pre-conception period, and they affect MDSCs as well as other immune cells that may be important for improving implantation success in women with RM.

Abstract Background: Every year, a large number of people worldwide suffer from gastrointestinal diseases such as diarrhea. Among this population, children under five are particularly vulnerable and are at increased risk of death or malnutrition. Enterotoxigenic Escherichia coli (ETEC) is a major cause of this severe diarrhea. The bacteria first adhere to intestinal cells using colonization factors (CFs), then produce heat-stable (ST) and heat-labile (LT) toxins, which together are responsible for causing the severe diarrheal illness.
Objective: to investigate the potential of the recombinant surface protein YncE, encapsulated within chitosan nanoparticles, as a novel vaccine candidate, and to evaluate its immunogenicity.
Methods: The recombinant YncE gene, encoding the surface protein YncE, was cloned, expressed, purified. Successful expression and purification were confirmed via Western blot analysis. The purified recombinant protein was subsequently encapsulated within chitosan nanoparticles, and the particle size was measured. BALB/c mice were divided into five groups: recombinant protein injection, nanoparticle injection, oral nanoparticle administration, combined oral-injection nanoparticle regimen, and control. Immunogenicity was evaluated by measuring antibody titers using an enzyme-linked immunosorbent assay (ELISA). Finally, a bacterial challenge assay was conducted on all immunized groups to evaluate protective efficacy.
Results: Following expression and purification, the size of the YncE-containing nanoparticles was measured. These nanoparticles successfully induced both humoral and mucosal immune responses. Furthermore, immunized mice challenged with the YncE-containing chitosan nanovaccine demonstrated protective immunity against ETEC.
Conclusion: Taken together, our results show that the YncE protein-when packaged in chitosan nanoparticles-protects mice against ETEC infection. This makes YncE a strong candidate for future vaccine development.

Abstract Background: Vitamin D plays a critical immunomodulatory role, and its deficiency has been consistently associated with the pathogenesis and activity of various autoimmune diseases. Despite high ambient sunlight in Southeast Iran, sociocultural factors and disease-related sun avoidance may increase hypovitaminosis D risk in patients with systemic lupus erythematosus (SLE).
Objective: To determine the prevalence of vitamin D deficiency among patients with SLE in Zahedan, Iran, and to evaluate its association with disease activity.
Methods: We conducted a cross-sectional study of 100 adult SLE patients at a major referral center. We measured disease activity using the Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K). Blood samples were tested for 25-hydroxyvitamin D [25(OH)D] levels using a chemiluminescence immunoassay. We grouped patients into three categories: deficient (below 20 ng/mL), insufficient (20–29.9 ng/mL), or sufficient (30 ng/mL or above). Data were analyzed using Spearman’s correlation and one-way ANOVA.
Results: The mean serum 25(OH)D level was 22.9 ± 12.2 ng/mL. Only 23% of patients exhibited sufficient vitamin D levels, whereas the remaining 77% demonstrated suboptimal levels (33% deficient, 44% insufficient). A significant inverse correlation was observed between serum 25(OH)D levels and SLEDAI-2K scores (r = -0.375, p<0.001). Furthermore, vitamin D levels demonstrated a graded decline with increasing disease severity; patients with high disease activity had significantly lower mean 25(OH)D concentrations compared to those with mild activity (11.95 ± 7.96 ng/mL vs. 25.18 ± 13.03 ng/mL; p=0.048).
Conclusion: Vitamin D deficiency is highly prevalent in SLE patients in Southeast Iran and is significantly associated with greater disease activity. Routine screening and further research into vitamin D supplementation are warranted.

Abstract Background: Cytokeratin 7 (CK7) and cytokeratin 20 (CK20) are widely used immunohistochemical (IHC) markers routinely employed to delineate the primary site of epithelial malignancies, especially when the primary tumor is unknown. However, access to high-quality commercial antibodies remains limited in many laboratory settings.
Objective: To establish a practical and reproducible workflow to generate mouse-derived polyclonal antibodies against human CK7 and CK20.
Methods: Recombinant full-length proteins and epitope-enriched fragments were expressed in Escherichia coli, purified, and used for immunization. Antibody responses were evaluated by ELISA, while specificity and diagnostic applicability were assessed using Western blot and IHC on formalin-fixed, paraffin-embedded tissue sections. Post-production refinement, including protein A purification and negative absorption, was applied to reduce non-specific binding and improve antibody performance.
Results: High-titer antibody responses were successfully generated, with epitope-based antigens eliciting stronger and more selective reactivity compared to full-length proteins. Initial antibody preparations showed broad staining across epithelial tissues, which is expected given the conserved nature of cytokeratin. However, purification and absorption steps significantly reduced non-specific staining and improved specificity. In IHC analyses, the generated antibodies demonstrated staining patterns consistent with established CK7/CK20 expression profiles and were comparable in interpretability to commercially available reagents.
Conclusion: This study demonstrates that diagnostically usable polyclonal antibodies against human CK7 and CK20 can be produced using a cost-effective and accessible approach. This workflow provides a practical alternative for laboratories with limited access to commercial antibodies and supports the continued use of IHC as a reliable tool for tumor classification.

Abstract

Abstract The manuscript underwent peer review by two reviewers. Following major revisions, the first reviewer recommended acceptance, and the abstract was submitted for PubMed indexing. The second reviewer’s delayed assessment identified substantial methodological and internal consistency issues. After evaluating these concerns, the Editor-in-Chief made the final decision to return the manuscript to the authors without further opportunity for revision and to withdraw it from publication. Accordingly, its status was to be changed to “Withdrawn” on the journal’s website and in PubMed.