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Background: Regulatory B cells (Bregs) play a pivotal role in suppressing immune responses, yet there is still a lack of cell surface markers that can rigorously identify them. In mouse models for multiple sclerosis (MS), TIM-1 or TIGIT expression on B cells is required for maintaining self-tolerance and regulating autoimmunity to the central nervous system. Here we investigated the activities of human memory B cells that differentially express TIM-1 and TIGIT to determine their potential regulatory function in healthy donors and patients with relapsing-remitting (RR) MS., Methods: FACS-sorted TIM-1+/-TIGIT+/- memory B (memB) cells co-cultured with allogenic CD4+ T cells were analyzed for proliferation and induction of inflammatory markers using flow cytometry and cytokine quantification, to determine Th1/Th17 cell differentiation. Transcriptional differences were assessed by SMARTSeq2 RNA sequencing analysis., Results: TIM-1-TIGIT- double negative (DN) memB cells strongly induce T cell proliferation and pro-inflammatory cytokine expression. The TIM-1+ memB cells enabled low levels of CD4+ T cell activation and gave rise to T cells that co-express IL-10 with IFNγ and IL-17A or FoxP3. T cells cultured with the TIM-1+TIGIT+ double positive (DP) memB cells exhibited reduced proliferation and IFNγ, IL-17A, TNFα, and GM-CSF expression, and exhibited strong regulation in Breg suppression assays. The functional activity suggests the DP memB cells are a bonafide Breg population. However, MS DP memB cells were less inhibitory than HC DP memB cells. A retrospective longitudinal study of anti-CD20 treated patients found that post-treatment DP memB cell frequency and absolute number were associated with response to therapy. Transcriptomic analyses indicated that the dysfunctional MS-derived DP memB/Breg population exhibited increased expression of genes associated with T cell activation and survival (CD80, ZNF10, PIK3CA), and had distinct gene expression compared to the TIGIT+ or TIM-1+ memB cells., Conclusion: These findings demonstrate that TIM-1/TIGIT expressing memory B cell subsets have distinct functionalities. Co-expression of TIM-1 and TIGIT defines a regulatory memory B cell subset that is functionally impaired in MS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Varghese, Kaskow, von Glehn, Case, Li, Julé, Berdan, Ho Sui, Hu, Krishnan, Chitnis, Kuchroo, Weiner and Baecher-Allan.)

The purpose of this study was to examine whether myeloid dendritic cells (mDCs) from patients with multiple sclerosis (MS) and healthy controls (HCs) become similarly tolerogenic when exposed to IL-27 as this may represent a potential mechanism of autoimmune dysregulation. Our study focused on natural mDCs that were isolated from HCs and MS patient peripheral blood mononuclear cells (PBMCs). After a 24-h treatment with IL-27 ± lipopolysaccharide (LPS), the mDCs were either harvested to identify IL-27-regulated gene expression or co-cultured with naive T-cells to measure how the treated DC affected T-cell proliferation and cytokine secretion. mDCs isolated from HCs but not untreated MS patients became functionally tolerogenic after IL-27 treatment. Although IL-27 induced both HC and untreated MS mDCs to produce similar amounts of IL-10, the tolerogenic HC mDCs expressed PD-L2, IDO1, and SOCS1, while the non-tolerogenic untreated MS mDCs expressed IDO1 and IL-6R. Cytokine and RNA analyses identified two signature blocks: the first identified genes associated with mDC tolerizing responses to IL-27, while the second was associated with the presence of MS. In contrast to mDCs from untreated MS patients, mDCs from HCs and IFNb-treated MS patients became tolerogenic in response to IL-27. The genes differentially expressed in the different donor IL-27-treated mDCs may contain targets that regulate mDC tolerogenic responses.

T cells are present in early stages of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and play a major role in disease outcome and long-lasting immunity. Nasal administration of a fully human anti-CD3 monoclonal antibody (Foralumab) reduced lung inflammation as well as serum IL-6 and C-reactive protein in moderate cases of COVID-19. Using serum proteomics and RNA-sequencing, we investigated the immune changes in patients treated with nasal Foralumab. In a randomized trial, mild to moderate COVID-19 outpatients received nasal Foralumab (100 μg/d) given for 10 consecutive days and were compared to patients that did not receive Foralumab. We found that naïve-like T cells were increased in Foralumab-treated subjects and NGK7 + effector T cells were reduced. CCL5, IL32, CST7, GZMH, GZMB, GZMA, PRF1 , and CCL4 gene expression were downregulated in T cells and CASP1 was downregulated in T cells, monocytes, and B cells in subjects treated with Foralumab. In addition to the downregulation of effector features, an increase in TGFB1 gene expression in cell types with known effector function was observed in Foralumab-treated subjects. We also found increased expression of GTP-binding gene GIMAP7 in subjects treated with Foralumab. Rho/ROCK1, a downstream pathway of GTPases signaling was downregulated in Foralumab-treated individuals. TGFB1, GIMAP7 , and NKG7 transcriptomic changes observed in Foralumab-treated COVID-19 subjects were also observed in healthy volunteers, MS subjects, and mice treated with nasal anti-CD3. Our findings demonstrate that nasal Foralumab modulates the inflammatory response in COVID-19 and provides a novel avenue to treat the disease.

Background: Parenteral anti-CD3 Mab (OKT3) has been used to treat transplant rejection and parental administration of a humanized anti-CD3 Mab (Teplizumab) showed positive effects in diabetes. Nasal administration of anti-CD3 Mab has not been carried out in humans. Nasal anti-CD3 Mab suppresses autoimmune diseases and central nervous system (CNS) inflammation in animal models. We investigated the safety and immune effects of a fully humanized, previously uncharacterized nasal anti-CD3 Mab (Foralumab) in humans and its in vitro stimulatory properties., Methods: In vitro , Foralumab were compared to UCHT1 anti-human CD3 mAb. For human administration, 27 healthy volunteers (9 per group) received nasal Foralumab or placebo at a dose of 10ug, 50ug, or 250ug daily for 5 days. Safety was assessed and immune parameters measured on day 1 (pre-treatment), 7, 14, and 30 by FACS and by scRNAseq., Results: In vitro , Foralumab preferentially induced CD8+ T cell stimulation, reduced CD4+ T cell proliferation and lowered expression of IFNg, IL-17 and TNFa. Foralumab induced LAP, TIGIT, and KLRG1 immune checkpoint molecules on CD8+ and CD4+ T cells in a mechanism independent of CD8 T cells. In vivo , nasal Foralumab did not modulate CD3 from the T cell surface at any dose. Immune effects were primarily observed at the 50ug dose and consisted of reduction of CD8+ effector memory cells, an increase in naive CD8+ and CD4+ T cells, and reduced CD8+ T cell granzyme B and perforin expression. Differentially expressed genes observed by scRNAseq in CD8+ and CD4+ populations promoted survival and were anti-inflammatory. In the CD8+ TEMRA population there was induction of TIGIT, TGFB1 and KIR3DL2, indicative of a regulatory phenotype. In the memory CD4+ population, there was induction of CTLA4, KLRG1, and TGFB whereas there was an induction of TGF-B1 in naïve CD4+ T cells. In monocytes, there was induction of genes (HLA-DP, HLA-DQ) that promote a less inflammatory immune response. No side effects were observed, and no subjects developed human anti-mouse antibodies., Conclusion: These findings demonstrate that nasal Foralumab is safe and immunologically active in humans and presents a new avenue for the treatment of autoimmune and CNS diseases., Competing Interests: WC was employed by Clementi, Ltd. HLW is chair of the scientific advisory board of Tiziana Life Sciences and received consulting fees and stock options from the company. KS is an employee of Tiziana Life Sciences. TC is a member of the scientific advisory board and serves as a consultant to Tiziana Life Sciences. CMB-A serves as a consultant to Tiziana Life Sciences. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Chitnis, Kaskow, Case, Hanus, Li, Varghese, Healy, Gauthier, Saraceno, Saxena, Lokhande, Moreira, Zurawski, Roditi, Bergmark, Giovannoni, Torti, Li, Quintana, Clementi, Shailubhai, Weiner and Baecher-Allan.)

Background: Immune hyperactivity is an important contributing factor to the morbidity and mortality of COVID-19 infection. Nasal administration of anti-CD3 monoclonal antibody downregulates hyperactive immune responses in animal models of autoimmunity through its immunomodulatory properties. We performed a randomized pilot study of fully-human nasal anti-CD3 (Foralumab) in patients with mild to moderate COVID-19 to determine if its immunomodulatory properties had ameliorating effects on disease., Methods: Thirty-nine outpatients with mild to moderate COVID-19 were recruited at Santa Casa de Misericordia de Santos in Sao Paulo State, Brazil. Patients were randomized to three cohorts: 1) Control, no Foralumab (n=16); 2) Nasal Foralumab (100ug/day) given for 10 consecutive days with 6 mg dexamethasone given on days 1-3 (n=11); and 3) Nasal Foralumab alone (100ug/day) given for 10 consecutive days (n=12). Patients continued standard of care medication., Results: We observed reduction of serum IL-6 and C-reactive protein in Foralumab alone vs . untreated or Foralumab/Dexa treated patients. More rapid clearance of lung infiltrates as measured by chest CT was observed in Foralumab and Foralumab/Dexa treated subjects vs . those that did not receive Foralumab. Foralumab treatment was well-tolerated with no severe adverse events., Conclusions: This pilot study suggests that nasal Foralumab is well tolerated and may be of benefit in treatment of immune hyperactivity and lung involvement in COVID-19 disease and that further studies are warranted., Competing Interests: JJ, VP and KS are employees of Tiziana, Life Sciences. HW is chair of the Scientific Advisory Board of Tiziana and received consulting fees and stock options from the company. TM and KM received consulting fees from the company. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Moreira, Matos, De Paula, Santana, Da Mata, Pansera, Cortina, Spinola, Keppeke, Jacob, Palejwala, Chen, Izzy, Healey, Rezende, Dedivitis, Shailubhai and Weiner.)

Defining and characterizing pathologies of the immune system requires precise and accurate quantification of abundances and functions of cellular subsets via cytometric studies. At this time, data analysis relies on manual gating, which is a major source of variability in large-scale studies. We devised an automated, user-guided method, X-Cyt, which specializes in rapidly and robustly identifying targeted populations of interest in large data sets. We first applied X-Cyt to quantify CD4(+) effector and central memory T cells in 236 samples, demonstrating high concordance with manual analysis (r = 0.91 and 0.95, respectively) and superior performance to other available methods. We then quantified the rare mucosal associated invariant T cell population in 35 samples, achieving manual concordance of 0.98. Finally we characterized the population dynamics of invariant natural killer T (iNKT) cells, a particularly rare peripheral lymphocyte, in 110 individuals by assaying 19 markers. We demonstrated that although iNKT cell numbers and marker expression are highly variable in the population, iNKT abundance correlates with sex and age, and the expression of phenotypic and functional markers correlates closely with CD4 expression.

Recent studies have demonstrated that the skin of a normal adult human contains 10-20 billion resident memory T cells, including various helper, cytotoxic, and regulatory T cell subsets, that are poised to respond to environmental antigens. Using only autologous human tissues, we report that both in vitro and in vivo, resting epidermal Langerhan cells (LCs) selectively and specifically induced the activation and proliferation of skin resident regulatory T (Treg) cells, a minor subset of skin resident memory T cells. In the presence of foreign pathogen, however, the same LCs activated and induced proliferation of effector memory T (Tem) cells and limited Treg cells' activation. These underappreciated properties of LCs, namely maintenance of tolerance in normal skin, and activation of protective skin resident memory T cells upon infectious challenge, help clarify the role of LCs in skin., (Copyright © 2012 Elsevier Inc. All rights reserved.)

CD4(+)CD25(high)CD127(low/-) forkhead box p3 (Foxp3)(+) regulatory T cells (T(reg) cells) possess functional plasticity. Here we describe a higher frequency of T helper type 1 (T(H)1)-like, interferon-γ (IFN-γ)-secreting Foxp3(+) T cells in untreated subjects with relapsing remitting multiple sclerosis (RRMS) as compared to healthy control individuals. In subjects treated with IFN-β, the frequency of IFN-γ(+)Foxp3(+) T cells is similar to that in healthy control subjects. In vitro, human T(reg) cells from healthy subjects acquire a T(H)1-like phenotype when cultured in the presence of interleukin-12 (IL-12). T(H)1-like T(reg) cells show reduced suppressive activity in vitro, which can partially be reversed by IFN-γ-specific antibodies or by removal of IL-12.

Studying the activity of homogeneous regulatory T cell (Treg) populations will advance our understanding of their mechanisms of action and their role in human disease. Although isolating human Tregs exhibiting low expression of CD127 markedly increases purity, the resulting Treg populations are still heterogeneous. To examine the complexity of the Tregs defined by the CD127 phenotype in comparison with the previously described CD4(+)CD25(hi) subpopulations, we subdivided the CD25(hi) population of memory Tregs into subsets based on expression of CD127 and HLA-DR. These subsets exhibited differences in suppressive capacity, ability to secrete IL-10 and IL-17, Foxp3 gene methylation, cellular senescence, and frequency in neonatal and adult blood. The mature, short telomere, effector CD127(lo)HLA-DR(+) cells most strongly suppressed effector T cells within 48 h, whereas the less mature CD127(lo)HLA-DR(-) cells required 96 h to reach full suppressive capacity. In contrast, whereas the CD127(+)HLA-DR(-) cells also suppressed proliferation of effector cells, they could alternate between suppression or secretion of IL-17 depending upon the stimulation signals. When isolated from patients with multiple sclerosis, both the nonmature and the effector subsets of memory CD127(lo) Tregs exhibited kinetically distinct defects in suppression that were evident with CD2 costimulation. These data demonstrate that natural and not induced Tregs are less suppressive in patients with multiple sclerosis.

CD4+CD25+ regulatory T cells (Treg) appear to be critical in regulating immune responses to self-antigens. Treg deficiency is associated with several human autoimmune diseases. Although substantial progress has been made in the study of murine and human Treg, their fundamental mechanism of action remains unknown. In this review, we discuss the phenotype of human natural Treg, their functional mechanism, and their role in autoimmune disease.

Regulatory T cells were initially identified and isolated in the mouse, by virtue of their endogenous expression of CD25 (IL-2R alphachain) and shown to inhibit both the in vivo development of autoimmunity and the in vitro proliferation of nonregulatory, CD4+CD25- T cells. In contrast to mouse cells, human regulatory T cells are not purified by isolating all CD25-expressing CD4 T cells ex vivo. Such cells can be isolated by targeting only the small percentage of human CD4 T cells that express high levels of CD25. This is best achieved by FACS sorting using the level of CD25 expressed on CD4- T cells to place the gate for discriminating high expression of CD25. This unit provides two widely used methods to isolate (FACS) or to enrich (magnetic beads) human CD4+CD25+ regulatory T cells from blood, along with an in vitro coculture assay to measure the anergic and suppressive features of human CD4+CD25+ regulatory T cells.

We describe a technology for generating recombinant polyclonal antibody libraries (PCALs) that enables the creation and perpetuation of standardized mixtures of polyclonal whole antibodies specific for a multiantigen (or polyantigen). Therefore, this technology combines the advantages of targeting multiple antigenic determinants -- high avidity, low likelihood of antigen 'escape variants', and efficient mediation of effector functions, with the advantages of using monoclonal antibodies -- unlimited supply of standardized reagents and the availability of the genetic material for desired manipulations. The technology for generating recombinant polyclonal antibody libraries begins with the creation of phage display Fab (antibody) libraries. This is followed by selection of sublibraries with desired antigen specificities, and mass transfer of the variable region gene pairs of the selected sublibraries to a mammalian expression vector for generation of libraries of polyclonal whole antibodies. We review here our experiments for selection of phage display antibody libraries against microbes and tumor cells, as well as the recent literature on the selection of phage display antibody libraries to multiantigen targets.

We have identified a new factor, CFX, in human serum and plasma that inhibits the growth of cultured human and mouse cell lines. CFX was determined to be a negatively charged, hydrophobic glycoprotein, with a native molecular weight of 110-120 kDa and a minimal active subunit of 55 kDa. It is precipitated by 60% ammonium sulfate and is resistant to heat treatment at 100 degrees C for 30 min. CFX was purified from human plasma to a single band on a gel which retained the cell growth inhibitory activity. Amino acid sequence analysis of the CFX band revealed sequences from four human glycoproteins, alpha1-antichymotrypsin, C1-esterase inhibitor, alpha1-antitrypsin, and alpha2-antiplasmin, all members of the superfamily of serpins. Of the four, C1-esterase inhibitor was shown to be the most potent cell growth inhibitor. These results suggest that serpins may play a cell growth inhibitory role in vivo, in addition to their role as protease inhibitors., (Copyright 2000 Academic Press.)

We had developed a technology for creation of recombinant polyclonal antibody libraries, standardized perpetual mixtures of polyclonal whole antibodies for which the genes are available and can be altered as desired. We report here the first phase of generating a polyclonal antibody library to Cryptosporidium parvum, a protozoan parasite that causes severe disease in AIDS patients, for which there is no effective treatment. BALB/c mice, immunized by neonatal oral infection with oocysts followed by intraperitoneal immunization with a sporozoite/oocyst preparation of C. parvum, were used for construction of a Fab phage display library in a specially-designed bidirectional vector. This library was selected for reactivity to an oocyst/sporozoite preparation, and was shown to be antigen-specific and diverse. Following mass transfer of the selected variable region gene pairs to appropriate mammalian expression vectors, such anti-C. parvum Fab phage display libraries could be used to develop chimeric polyclonal antibody libraries, with mouse variable regions and human constant regions, for passive immunotherapy of C. parvum infection.

Epithelial repair following acute lung injury involves proliferation and differentiation of existing Clara cells and type II cells. Other mechanisms of epithelial repair may be involved in particularly severe cases. We used epithelial cell-specific markers to examine changes in the mouse lung epithelium 28 days after bleomycin treatment. The spatial distribution of surfactant proteins A, B, C (SPA, SPB, SPC), and Clara cell-specific protein (CC10) mRNA was compared by in situ hybridization in serial lung sections. CC10 mRNA-containing airway cells were replaced in many areas by SPB mRNA-expressing, ciliated cells that did not contain CC10 mRNA. In distal airway regions, we observed a subpopulation of epithelial cells that appeared to express SPA, SPB, SPC, and CC10 mRNA, and speculated that they may represent a multipotential stem cell population. These cells were found in focal clusters, which suggests that they expanded from a common cell. CC10 mRNA-containing cells were seen in alveolar-like structures thought to be the result of Clara cell migration or outpocketing. Our data suggest that there are repair mechanisms involved in epithelial repair after severe injury that have not previously been described.

The contribution of T-cell-receptor beta-chain diversity to the T-cell antigen-specific repertoire was investigated using single-chain T-cell-receptor transgenic mice. Animals that express the rearranged beta-chain gene from a T hybridoma with specificity for a hen egg lysozyme peptide, designated HEL (85-96) were analysed for their ability to respond to a panel of diverse antigens. Transgenic mice exhibited a significantly elevated response to HEL (85-96) which was shown to be due to an increased frequency of HEL (85-96)-specific T-cell progenitors. This increased frequency of specific progenitors resulted in the ability of transgenic mice to respond to the peptide in the absence of antigen priming. Conversely, transgenic mice failed to respond to any other antigen tested. Furthermore, this apparent deficiency was associated with a significant decrease in the frequency of antigen-specific T-cell progenitors in transgenic mice. Surprisingly, the ability to launch an alloresponse was unaffected by the exclusive expression of the transgene-derived beta-chain. These results indicate that beta-chain diversity is crucial for the ability of the T-cell population to elicit a rapid and robust response to the profusion of different antigen/major histocompatibility complex (MHC) ligands potentially encountered by an individual. Furthermore, these results suggest a lesser role for beta-chain diversity in contributing to allorecognition, and support a model in which the direct recognition of peptide-mediated conformational MHC forms is the major contributor to the alloreactive response exhibited by the majority of T cells.

Clinical use of the antineoplastic agent bleomycin is restricted due to pulmonary toxicity. Murine models of bleomycin-induced pulmonary fibrosis have been developed in an attempt to understand the mechanisms involved in the fibrotic process. Studies have shown that the alveolar epithelium is damaged early after bleomycin treatment. The purpose of this study was to evaluate the pattern of gene expression in airway and alveolar epithelial cells after bleomycin exposure in mice that vary in susceptibility to bleomycin-induced fibrosis. Surfactant protein C (SPC) and Clara cell-specific protein (CC10) mRNA were used as cell-specific markers of alveolar type II cells and airway Clara cells, respectively. Mice were treated with a single intratracheal dose of bleomycin and the pattern of SPC and CC10 transcripts was examined by in situ hybridization. The pattern of SPC mRNA 28 days after treatment was uniform in controls and resistant mice but exhibited a patchy appearance in sensitive mice. Bleomycin treatment also resulted in a strain-dependent loss of CC10 mRNA-expressing cells. In sensitive mice 28 days after treatment, SPC mRNA was ectopically expressed in the distal bronchiolar epithelium in a morphologically distinct cell type. Serial sections revealed that these cells either coexpressed CC10 mRNA or were located adjacent to CC10 mRNA-containing cells. This unique cell population may represent a progenitor cell type important in epithelial repair. The strain-dependent changes in CC10 and SPC gene expression after bleomycin treatment are suggestive of a role for the epithelium in pulmonary fibrosis versus repair.

Susceptibility of mice to the induction of pulmonary fibrosis by bleomycin sulfate is inbred strain dependent, with C57BL/6 mice exhibiting high sensitivity to the drug and BALB/c mice demonstrating a resistant phenotype. The lungs of bleomycin treated C57BL/6J and BALB/cBy mice were analyzed for their mRNA expression level of a panel of cytokines using a semi-quantitative polymerase chain reaction (SQ-PCR) assay. Transforming growth factor-beta 1 (TGF-beta 1) mRNA was found to increase sevenfold by 5 days after bleomycin treatment of C57BL/6J (sensitive) mice. BALB/cBy (resistant) animals demonstrated a lower level of TGF-beta 1 mRNA induction, approximately threefold, after bleomycin administration. Analysis of interleukin-1 beta (IL-1 beta) mRNA levels also revealed a difference between the two strains, with BALB/cBy mice expressing approximately fourfold higher IL-1 beta mRNA levels than C57BL/6J mice. This result suggested possible protection by IL-1 beta. Analysis of (C57BL/6JxBALB/cBy)F1 hybrids, which are shown in this report to be sensitive to bleomycin-induced fibrosis, revealed a high IL-1 beta mRNA level, similar to that in the resistant parent. Thus, the observed strain variation in the level of IL-1 beta mRNA is not associated with differences in susceptibility to the induction of pulmonary fibrosis. In contrast, strain variation in interleukin-6 (IL-6) mRNA levels was observed that was completely concordant with the segregation of susceptibility phenotypes between the parental and F1 strains. This result indicates a possible association between sensitivity to bleomycin-induced fibrosis and inducibility of IL-6 mRNA upon drug treatment. Analysis of TGF-beta 2, interferon-gamma, interleukin-2, interleukin-3, and interleukin-4 (IL-4) mRNA showed no detectable strain variation in steady state mRNA levels in the lung as a consequence of bleomycin treatment. In contrast, the level of IL-4 receptor mRNA was induced to a higher degree in both sensitive groups (C57BL/6J and F1) than in resistant mice (BALB/cBy). Therefore, modulation of the IL-4 response, not at the level of IL-4 but through regulation of the IL-4 receptor, may play a role in pulmonary fibrogenesis.

Ly-48 is a major sialoglycoprotein expressed on the surface of a variety of mouse hematopoietic cells that exhibits many characteristic isoforms and may function in signal transduction and cell adhesion. Ly-48 is recognized by the 3E8-specific monoclonal antibody (mAb) and it has been suggested that it is the same antigen recognized by another mAb known as S7. In this report, we demonstrate definitively by transfection of a Ly-48 cDNA that S7 and two previously uncharacterized mAbs, S11 and S15, recognize the same antigen as the 3E8-specific mAb. However, 2-D gel immunoblot analyses demonstrate the complex nature of Ly-48. Although all four mAbs react similarly with lysates from the M-45 B-cell myeloma line, 2-D immunoblot analyses of the EL-4 T-cell line reveal three distinct patterns of reactivity. Further, while transfection of Ly-48 into the K562 erythroleukemic cell line conferred reactivity to all four mAbs, transfection of the Ly-48 cDNA into the nonhematopoietic cell line, Line 1, conferred reactivity only to the S11 and S15 mAbs. Thus, the Line 1 transfectants suggest the importance of posttranslational modifications in the expression of the 3E8 and S7 epitopes. Interestingly, developing fetal liver cells show the same pattern of differential Ly-48-specific mAb reactivity. The developing early fetal liver cells are reactive with S11 and S15 but are negative, to very weakly, reactive with the 3E8- and S7-specific mAbs. These results show that Ly-48 epitopes can be expressed independently on cell lines in vitro and are differentially expressed on healthy cells in vivo.