Overall survival after reduced-intensity conditioning (RIC) allogeneic hematopoietic cell transplantation (HCT) using alemtuzumab, fludarabine, and melphalan is associated with high rates of mixed chimerism (MC) and secondary graft failure (GF). We hypothesized that peritransplantation alemtuzumab levels or specific patterns of inflammation would predict these risks. We assessed samples from the Bone Marrow Transplant Clinical Trials Network 1204 (NCT01998633) to study the impact of alemtuzumab levels and cytokine patterns on MC and impending or established secondary GF (defined as donor chimerism <5% after initial engraftment and/or requirement of cellular intervention). Thirty-three patients with hemophagocytic lymphohistiocytosis (n = 25) and other IEIs (n = 8) who underwent HCTs with T-cell-replete grafts were included. Patients with day 0 alemtuzumab levels ≤0.32 μg/mL had a markedly lower incidence of MC, 14.3%, vs 90.9% in patients with levels >0.32 μg/mL (P = .008). Impending or established secondary GF was only observed in patients with day 0 alemtuzumab levels >0.32 μg/mL (P = .08). Unexpectedly, patients with impending or established secondary GF had lower CXCL9 levels. The cumulative incidence of impending or established secondary GF in patients with a day 14+ CXCL9 level ≤2394 pg/mL (day 14+ median) was 73.6% vs 0% in patients with a level >2394 pg/mL (P = .002). CXCL9 levels inversely correlated with alemtuzumab levels. These data suggest a model in which higher levels of alemtuzumab at day 0 deplete donor T cells, inhibit the graft-versus-marrow reaction (thereby suppressing CXCL9 levels), and adversely affect sustained engraftment in the nonmyeloablative HCT setting. This trial was registered at www.clinicaltrials.gov as #NCT01998633.

Neurodegenerative diseases (ND) are characterized by progressive loss of neuronal function. Mechanisms of ND pathogenesis are incompletely understood, hampering the development of effective therapies. Langer-hans cell histiocytosis (LCH) is an inflammatory neoplastic disorder caused by hematopoietic progenitors ex-pressing mitogen-activated protein kinase (MAPK)-activating mutations that differentiate into senescent myeloid cells that drive lesion formation. Some individuals with LCH subsequently develop progressive and incurable neurodegeneration (LCH-ND). Here, we showed that LCH-ND was caused by myeloid cells that were clonal with peripheral LCH cells. Circulating BRAFV600E+ myeloid cells caused the breakdown of the blood-brain barrier (BBB), enhancing migration into the brain parenchyma where they differentiated into senescent, inflammatory CD11a+ macrophages that accumulated in the brainstem and cerebellum. Blocking MAPK activity and senescence programs reduced peripheral inflammation, brain parenchymal infil-tration, neuroinflammation, neuronal damage and improved neurological outcome in preclinical LCH-ND. MAPK activation and senescence programs in circulating myeloid cells represent targetable mechanisms of LCH-ND.

Neurodegenerative diseases (ND) are characterized by progressive loss of neuronal function. Mechanisms of ND pathogenesis are incompletely understood, hampering the development of effective therapies. Langerhans cell histiocytosis (LCH) is an inflammatory neoplastic disorder caused by hematopoietic progenitors expressing MAPK activating mutations that differentiate into senescent myeloid cells that drive lesion formation. Some patients with LCH subsequently develop progressive and incurable neurodegeneration (LCH-ND). Here, we show that LCH-ND is caused by myeloid cells that are clonal with peripheral LCH cells. We discovered that circulating BRAF V600E + myeloid cells cause the breakdown of the blood-brain barrier (BBB), enhancing migration into the brain parenchyma where they differentiate into senescent, inflammatory CD11a + macrophages that accumulate in the brainstem and cerebellum. Blocking MAPK activity and senescence programs reduced parenchymal infiltration, neuroinflammation, neuronal damage and improved neurological outcome in preclinical LCH-ND. MAPK activation and senescence programs in circulating myeloid cells represent novel and targetable mechanisms of ND.

The ability of chimeric antigen receptor (CAR) T cells to initiate and sustain an effective immune response entails a delicate balance between T-cell activation and deactivation and is key for complete tumor elimination. CD6 is a co-receptor that fine-tunes T cell receptor signal strength through a unique dual function intracellular domain. CD6 is alternatively spliced, creating isoforms that lack distinct regions which interact with activating or inhibitory adapter proteins, thereby delivering inhibitory or activating signals, respectively. We hypothesized that CAR T cell fitness, namely their ability to sustain their effector function, can be achieved by using alternatively spliced CD6 isoforms which lack exons responsible for inhibitory signaling. First, we discovered that overexpression of the canonical full-length CD6 molecule, isoform CD6A, resulted in marginal improvement of CAR T cell function, while its deletion using CRISPR/Cas9 depressed the antitumor CAR T cell functionality. This supported a net stimulatory role for CD6 in CAR T cell function. To study their effect on cellular fitness, we then synthesized and overexpressed known alternatively spliced cytoplasmic isoforms CD6B, CD6C, CD6D, CD6E, and CD6F on CAR T cells and, noted a significant tendency towards preserving the CAR T cell naïve and central memory compartments, upon tumor encounter. Overexpression of isoform CD6F, which lacks exon 9, and to a lesser extent isoforms CD6C and CD6E enhanced the short- and long-term cytotoxicity of HER2 and CD19 targeting CAR T cells when compared to CAR T cells alone or CAR T cells overexpressing, CD6A. Despite exhibiting a lower proliferation index, and lower CAR CD3ζ phosphorylation at rest, CD6F CAR T cells secreted significantly higher concentrations of the Th1 cytokine, IFN-γ, and mediated more sustained in vitro cytotoxicity long-term, with more complete elimination of large tumor cell loads. Lastly, in an orthotopic model of human glioblastoma, the adoptive transfer of CD6F HER2 CAR T cells induced a more significant but also more sustained anti-glioma effect that translated into a significant survival advantage for experimental animals. Our data support that the overexpression of the alternatively spliced CD6 isoforms improves CAR T cell fitness resulting in superior antitumor activity both in vitro and in vivo. Citation Format: Jessica S. Morris, Ahmed Z. Gad, Lea M. Godret-Miertschin, Ryan Fleischmann, Vita Salsman, Sujith K. Joseph, Nabil Ahmed. CD6 isoforms improve CAR T cell fitness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1151.

Langerhans cell histiocytosis (LCH) is a myeloid neoplastic disorder characterized by granulomatous lesions with CD207+ dendritic cells associated with a wide spectrum of clinical presentations. Activating MAPK pathway genes (most frequently BRAFV600E) are identified in lesion DCs as well as in precursor cells in bone marrow and peripheral blood in some cases. LCH typically arises in children, but can also occur de novo in adults. Erdheim-Chester disease (ECD) is a related histiocytic disorder characterized by CD163+ histiocytic lesions with activating MAPK mutations (most frequently BRAFV600E) arising primarily in adults. Unlike LCH, ECD lesion distribution frequently includes kidneys and heart. In order to test the impact of cell of origin on disease phenotype, we developed Cre-inducible mouse models in which BRAFV600E expression is enforced at distinct stages of differentiation: BRAFV600ECD11c and BRAFV600EMSaA3. ITGAX/CD11c is expressed across a range of myeloid dendritic cell and monocyte lineages, where MS4a3 is expressed exclusively in GMPs and GMP-derived cells (e.g. monocytes). BRAFV600E expression was enforced through Cre-mediated recombination to create BRAFV600ECD11c and BRAFV600EMSaA3 mice. Mice were analyzed at weeks 5, 10, 25, and 52 for body and organ weights and histology. Tissues were characterized by immunohistochemistry, gene expression, Luminex, CYTOF and RNASeq. BRAFV600ECD11c mice exhibited a more aggressive phenotype than BRAFV600EMS4A3 counterparts, though both developed systemic histiocytic infiltration. Kinetics of organomegaly, histiocytic infiltration, and systemic inflammation was much slower in the BRAFV600EMS4a3 mice. Notably, BRAFV600EMS4a3 demonstrated increased enrichment of CD207-negative macrophages in spleen, liver and bone marrow. BRAFV600EMS4a3 mice readily survived past 52 weeks, while BRAFV600ECD11c had 100% mortality by 16 weeks. Bulk RNASeq and Ingenuity Pathway Analysis identified relative decrease in macrophage functionality in BRAFV600EMSaA3 mice. Additionally, macrophage infiltrate was observed in heart, major blood vessels and kidneys of BRAFV600EMS4a3 where these organs were not affected in BRAFV600ECD11c. The same mutation that creates an aggressive LCH-like phenotype in CD11c-derived cells creates an indolent ECD-like phenotype in monocyte-derived cells. This study demonstrates a critical role for ontogeny in pathogenesis of histiocytic disorders. Further, the BRAFV600EMS4a3 mouse provides an ECD model for pre-clinical testing of therapeutic strategies.

Abstract Introduction The BMT-CTN 1204 study for Hemophagocytic Syndromes or Selected Primary Immune Deficiencies (NCT01998633) (RICHI) was a single arm study testing safety and efficacy of reduced intensity conditioning (RIC) with alemtuzumab (1mg/kg), fludarabine (150 mg/m2) and melphalan (140 mg/m2). Survival was favorable compared to historical studies, but patients experienced high rates of mixed chimerism (MC) and ultimate secondary graft failure (GF). Mechanisms for GF are not known. Expansion of recipient T cells and interferon-gamma pathway activation have been proposed as drivers for GF. However, high peri-transplant alemtuzumab levels have been associated with higher risk of MC and eventual secondary GF, suggesting an inverse relationship between GF and immune activation in the context of RIC. In order to elucidate mechanisms of GF for patients on the RICHI study, we systematically evaluated cytokine patterns and alemtuzumab levels and their association with durable engraftment. Methods Serial blood samples were collected, processed, and stored for consenting patients at day -14 (window: day -28 to -14), day -7 (+/- 1 day), day -1 (+/- 1), day +1 (+1 to +3), day +14 (+/- 2), day +28 (+/- 2), day +42 (+/- 3), day +70 (+/- 10), and day +100 (+/- 10). Alemtuzumab levels were measured using a flow cytometric assay as previously described. Comprehensive cytokine analysis was performed for over 100 analytes using the MagPix platform. Primary GF was defined as donor chimerism <5% prior to day +42. Secondary GF was defined as donor chimerism <5% after initial engraftment and/or requirement of donor lymphocyte infusion (DLI) or second HCT (with or without conditioning) to manage MC or graft loss. Mixed chimerism (MC) was defined as donor chimerism <95% on at least one occasion. Results Thirty-three patients were included in this study with HLH (n=25), CAEBV (n=3), CGD (n=2), HIGM (n=2), and IPEX (n=1). All patients received bone marrow grafts and 27 (82%) patients had unrelated donors. Twenty-one grafts were 8/8 or 6/6 HLA-matched (64%) and 12 grafts were 7/8 HLA-matched (36%). Among all patients, 1 patient (3%) developed primary GF, 22 (67%) developed mixed chimerism (MC), and 11 patients (33%) developed secondary GF. Survival with sustained engraftment without DLI or second HCT was 40.0%. We first evaluated peripheral blood levels of 100+ cytokines. Analysis revealed significant differences between patients with and without GF as shown in Figure 1A. Notably, on day +14 and +28, patients with secondary GF had significantly lower CXCL9 levels than those without GF. We then estimated the cumulative incidence (CI) of secondary GF among patients with CXCL9 levels above and below the day +14 median level of 2394pg/mL. The CI of secondary GF in patients with a day +14 CXCL9 level ≤2394pg/mL was 73.6% vs 0% in patients with a level >2394pg/mL (p=0.002). The CI of secondary GF in patients with a day +28 CXCL9 level ≤2867pg/mL (day +28 median) was 64.3%, vs 0% in patients with levels >2867pg/mL (p=0.004). We then sought to correlate CXCL9 levels with alemtuzumab exposure, as high alemtuzumab levels would result in more efficient T cell depletion of donor grafts that could lead to lower CXCL9 levels. Indeed, CXCL9 levels inversely correlated with day 0 alemtuzumab levels. Patients with day 0 alemtuzumab levels >0.32µg/mL had lower CXCL9 levels compared to patients with levels ≤0.32µg/mL (Figure 1B). Finally, we examined the impact of alemtuzumab levels on MC and secondary GF. Patients with day 0 alemtuzumab levels ≤0.32µg/mL had a lower CI of MC compared to patients with levels >0.32µg/mL, 14.3% vs 90.9%, respectively (p=0.03). The CI of secondary GF was 0% in patients with day 0 alemtuzumab levels ≤0.32µg/mL compared to 54.3% in patients with levels >0.32µg/mL (p=0.08). Conclusions This study demonstrates a strong relationship between alemtuzumab levels and durable engraftment. Further, interferon gamma activity, as reflected by CXCL9, inversely correlates with peri-transplant alemtuzumab levels in this prospective cohort treated with RIC. Our findings support the paradigm that higher alemtuzumab levels drive efficient T cell depletion of the stem cell product which increases the risk of MC and secondary GF, suggesting that donor T cells promote engraftment via a graft versus hematopoiesis function. Precision alemtuzumab dosing strategies may offer an opportunity to improve outcomes for patients who undergo RIC HCT. Figure 1 Figure 1. Disclosures Pulsipher: Adaptive: Research Funding; Equillium: Membership on an entity's Board of Directors or advisory committees; Jasper Therapeutics: Honoraria. Bollard: Neximmune: Current equity holder in publicly-traded company; Catamaran Bio: Membership on an entity's Board of Directors or advisory committees; Cabaletta Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Mana Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; Cellectis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Repertoire Immune Medicines: Current equity holder in publicly-traded company; ROCHE: Consultancy, Honoraria; SOBI: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kean: Regeneron: Research Funding; Bristol Myers Squibb: Patents & Royalties: From clinical trial data, Research Funding; Bluebird Bio: Research Funding; Gilead: Research Funding; Vertex: Consultancy; Novartis: Consultancy; EMD Serono: Consultancy. Jordan: Sobi: Consultancy. Allen: Sobi: Consultancy. OffLabel Disclosure: Alemtuzumab, humanized monoclonal antibody against CD52, used as part of allogeneic HCT conditioning

Key points Impaired growth during fetal life can reprogramme heart development and increase the risk for long‐term cardiovascular dysfunction. It is uncertain if the developmental window during which the heart is vulnerable to reprogramming as a result of inadequate nutrition extends into the postnatal period. We found that adult female mice that had been undernourished only from birth to 3 weeks of age had disproportionately smaller hearts compared to males, with thinner ventricle walls and more mononucleated cardiomyocytes. In females, but not males, cardiac diastolic function, and heart rate responsiveness to adrenergic stimulation were limited and maximal exercise capacity was compromised. These data suggest that the developmental window during which the heart is vulnerable to reprogramming by inadequacies in nutrient intake may extend into postnatal life and such individuals could be at increased risk for a cardiac event as a result of strenuous exercise. AbstractAdults who experienced undernutrition during critical windows of development are at increased risk for cardiovascular disease. The contribution of cardiac function to this increased disease risk is uncertain. We evaluated the effect of a short episode of postnatal undernutrition on cardiovascular function in mice at the whole animal, organ, and cellular levels. Pups born to control mouse dams were suckled from birth to postnatal day (PN) 21 on dams fed either a control (20% protein) or a low protein (8% protein) isocaloric diet. After PN21 offspring were fed the same control diet until adulthood. At PN70 was measured by treadmill test. At PN80 cardiac function was evaluated by echocardiography and Doppler analysis at rest and following β‐adrenergic stimulation. Isolated cardiomyocyte nucleation and Ca2+ transients (with and without β‐adrenergic stimulation) were measured at PN90. Female mice that were undernourished and then refed (PUN), unlike male mice, had disproportionately smaller hearts and their exercise capacity, cardiac diastolic function, and heart rate responsiveness to adrenergic stimulation were limited. A reduced left ventricular end diastolic volume, impaired early filling, and decreased stored energy at the beginning of diastole contributed to these impairments. Female PUN mice had more mononucleated cardiomyocytes; under resting conditions binucleated cells had a functional profile suggestive of increased basal adrenergic activation. Thus, a brief episode of early postnatal undernutrition in the mouse can produce persistent changes to cardiac structure and function that limit exercise/functional capacity and thereby increase the risk for the development of a wide variety of cardiovascular morbidities.

Background: Rapid growth of skeletal muscle in the neonate requires the coordination of protein deposition and myonuclear accretion. During this developmental stage, muscle protein synthesis is highly sensitive to amino acid supply, especially Leu, but we do not know if this is true for satellite cells, the source of muscle fiber myonuclei. Objective: We examined whether dietary protein restriction reduces myonuclear accretion in the neonatal pig, and if any reduction in myonuclear accretion is mitigated by restoring Leu intake. Methods: Neonatal pigs (1.53 +/- 0.2 kg) were fitted with jugular vein and gastric catheters and fed 1 of 3 isoenergetic milk replacers every 4 h for 21 d: high protein [HP; 22.5 g protein/(kg/d); n = 8]; restricted protein [RP; 11.2 g protein/(kg/d); n = 10]; or restricted protein with Leu [RPL; 12.0 g protein/(kg/d); n = 10]. Pigs were administered 5-bromo-2'deoxyuridine (BrdU; 15 mg/kg) intravenously every 12 h from days 6 to 8. Blood was sampled on days 6 and 21 to measure plasma Leu concentrations. On day 21, pigs were killed and the longissimus dorsi (LD) muscle was collected to measure cell morphometry, satellite cell abundance, myonuclear accretion, and insulin-like growth factor (IGF) system expression. Results: Compared with HP pigs, postprandial plasma Leu concentration in RP pigs was 37% and 47% lower on days 6 and 21, respectively (P < 0.05); Leu supplementation in RPL pigs restored postprandial Leu to HP concentrations. Dietary protein restriction reduced LD myofiber cross-sectional area by 21%, satellite cell abundance by 35%, and BrdU+ myonuclear abundance by 25% (P < 0.05); Leu did not reverse these outcomes. Dietary protein restriction reduced LD muscle IGF2 expression by 60%, but not IGF1 or IGF1R expression (P < 0.05); Leu did not rescue IGF2 expression. Conclusions: Satellite cell abundance and myonuclear accretion in neonatal pigs are compromised when dietary protein intake is restricted and are not restored with Leu supplementation.

Perinatal skeletal muscle growth rates are a function of protein and myonuclear accretion. Precocious exposure of the fetus to glucocorticoids (GLC) in utero impairs muscle growth. Reduced muscle protein synthesis rates contribute to this response, but the consequences for myonuclear hyperplasia are unknown. To test the hypothesis that blunting of Pax7+ muscle progenitor cell proliferative activity by GLC in vivo also contributes to reduced fetal muscle growth, pregnant rats were administered dexamethasone (DEX: 1 mg/L drinking water) from embryonic day (ED) 13 to ED21. Their responses were compared to pair-fed (PF) and ad libitum-fed controls (CON). Bromodeoxyuridine (BrdU) was administered before delivery to measure myonuclear accretion. Fetal hind limb and diaphragm muscles were collected at term and analyzed for myofiber cross-sectional area (CSA), total and BrdU+ myonuclei, Pax7+ nuclei, MyoD and myogenin protein and mRNA abundance and myosin heavy chain (MyHC) isoform composition. Mean fiber CSA, myonuclei/myofiber and Pax7+ nuclei/myofiber ratios were reduced in DEX compared to those in CON and PF muscles; CSA/myonucleus, BrdU+/total myonuclei and BrdU+ myonuclei/Pax7+ nuclei were similar among groups. Myogenin abundance was reduced and MyHC-slow was increased in DEX fetuses. The data are consistent with GLC inhibition of muscle progenitor cell proliferation limiting satellite cell and myonuclear accretion. The response of PF-fed compared to CON muscles indicated that decreased food consumption by DEX dams contributed to the smaller myofiber CSA but did not affect Pax7+ nuclear accretion. Thus, the effect on satellite cell reserve and myonuclear number also contributes to the blunting of fetal muscle growth by GLC.

Undernutrition during development increases the risk for adult cardiovascular disease. To determine if PUN alters heart structure function, and maximal exercise capacity in adulthood, newborn control mouse pups were fostered to dams fed a control (CON; 20% protein) or a protein-restricted diet (PUN; 8% protein; n=7-10 litters/treatment). At 21 d (PN21) pups were fed the CON diet ad lib. At PN70 a maximal treadmill test was performed to determine VO2peak; maximal vertical work was calculated for the final 30 sec. At PN80 cardiac function was evaluated by echocardiography. VO2peak was the same in CON and PUN mice and higher in males than females (249 + 9 and 169 + 6 ml/hr, respectively, P<0.001); gender x diet, NS. Maximal vertical work at VO2peak was higher in CON than PUN females (1.64 + 0.07 and 1.33 + 0.06 J/min, respectively, P < 0.01) but not males; gender x diet, P<0.02. Resting cardiac output was higher in CON than PUN offspring (18.8 + 0.7 and 16.3 + 0.7 ml/min, respectively; P<0.02) and in males than females (19.2 + 0.7 and 15.9 + 0.7 ml/min, respectively; P<0.01; gender x diet, NS) due to differences in stroke volume (CON: 43.0 ± 1.5; PUN: 37.7 ± 1.4 µl; P<0.05; males: 46.8 ± 1.5; females: 34.0 ± 1.4 µl; P<0.05), and left ventricular mass (CON: 104 ± 5; PUN: 90 ± 5 mg; P<0.05; males: 108 ± 5; females: 90 ± 5 mg; P<0.05). Thus, PUN permanently reduces left ventricular mass which in turn reduces stroke volume and maximal treadmill work capacity in female mice. Male mice were able to compensate for the reduction in cardiac output and maintain maximal exercise capacity. Supported by NIH AR46308, T32HL07676, USDA/ARS 6250-51000-051

Key points Inadequate nutrient intake during early life can programme a low adult muscle mass. We have used a mouse model to identify the developmental window when the skeletal musculature is vulnerable to programming and to identify factors that limit the muscle's ability to respond when normal nutrition is restored. We established that the developmental age when nutritional rehabilitation occurs following an episode of poor nutrition, rather than the duration or severity of the nutrient restriction, is the critical factor that determines if muscle mass can be recuperated. The ability to recover depends on whether the muscles’ translational capacity, i.e. ribosomal abundance, can increase sufficiently to raise protein synthesis rates sufficiently to accelerate protein deposition. We show that the ability to increase ribosomal abundance was associated with increased expression of the nucleolar transcription factor UBF (upstream binding factor), which regulates RNA polymerase 1 activity and rRNA transcription, the limiting factor for ribosomal production. AbstractNutritionally‐induced growth faltering in the perinatal period has been associated with reduced adult skeletal muscle mass; however, the mechanisms responsible for this are unclear. To identify the factors that determine the recuperative capacity of muscle mass, we studied offspring of FVB mouse dams fed a protein‐restricted diet during gestation (GLP) or pups suckled from postnatal day 1 (PN1) to PN11 (E‐UN), or PN11 to PN22 (L‐UN) on protein‐restricted or control dams. All pups were refed under control conditions following the episode of undernutrition. Before refeeding, and 2, 7 and 21 days later, muscle protein synthesis was measured in vivo. There were no long‐term deficits in protein mass in GLP and E‐UN offspring, but in L‐UN offspring muscle protein mass remained significantly smaller even after 18 months (P < 0.001). E‐UN differed from L‐UN offspring by their capacity to upregulate postprandial muscle protein synthesis when refed (P < 0.001), a difference that was attributable to a transient increase in ribosomal abundance, i.e. translational capacity, in E‐UN offspring (P < 0.05); translational efficiency was similar across dietary treatments. The postprandial phosphorylation of Akt and extracellular signal‐regulated protein kinases were similar among treatments. However, activation of the ribosomal S6 kinase 1 via mTOR (P < 0.02), and total upstream binding factor abundance were significantly greater in E‐UN than L‐UN offspring (P < 0.02). The results indicate that the capacity of muscles to recover following perinatal undernutrition depends on developmental age as this establishes whether ribosome abundance can be enhanced sufficiently to promote the protein synthesis rates required to accelerate protein deposition for catch‐up growth.

We investigated if undernutrition (UN) in the neonatal period of the mouse has any long‐term impact on activity following nutritional rehabilitation. Newborn pups born to control dams were cross‐fostered to dams fed either a low protein (8 % casein) or a control (CON; 20% casein) diet. On postnatal (PN) d 22 female offspring (n=18–20/diet) were weaned to the CON diet and individually housed in metabolic cages for measurement of energy expenditure, food intake, and ambulatory cage activity until PN43; half the mice also had access to free running wheels (FW). Body wt and composition (by QMR) were measured at PN22 and PN43. At PN22 and PN43, body wt of UN pups were 75% and 88% those of CON (P<0.001) and not alterd by FW. Lean gain as % body wt gain was greater in FW than sedentary (SED) pups and similar in UN and CON mice. Daily running distance increased with age, but UN pups ran only 46% the distance of CON pups (P<0.001); the difference was greater at PN43 than PN22 (P<0.02). Ambulatory activity was unaffec ted by preweaning UN and was greater in FW than SED pups (P<0.01). Differences in food intake and energetic efficiency could not account for the difference in FW running between groups. UN in early life reduces voluntary running, but not ambulatory, activity, and this response is not reversible upon nutritional rehabilitation. (Supported by NIH AR46308 and USDA/ARS 6250–51000‐054)