Early-life exposures to specific commensal microbes restrains autoimmunity

Abstract

The present disclosure is directed to the development of an early-life microbiome consortium that, when administered to young subjects, can protect the subject from, reduce the risk of, and / or reduce the severity of autoimmunity.

Description

[0001] DESCRIPTION

[0002] EARLY-LIFE EXPOSURES TO SPECIFIC COMMENSAL MICROBES RESTRAINS AUTOIMMUNITY PRIORITY CLAIM

[0003] This application claims benefit of priority to U.S. Provisional Application Serial No.

[0004] 63 / 670,367, filed February 19, 2025, the entire contents of which are hereby incorporated by reference.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0006] This invention was made with government support under grant no. DK 1 3453 and grant no. AI146629 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0007] BACKGROUND

[0008] 1. Field of the Disclosure

[0009] The present disclosure relates generally to the fields of immunology and microbiology. More particularly, the disclosure relates to the gut microbiome and its effects on early development of immune function and protection from autoimmunity.

[0010] 2. Background

[0011] In humans, early-life perturbations of key host-microbe interactions, particularly during the first 100 days of life, are associated with an increased risk of diseases such as allergy, asthma, psoriasis, obesity, and inflammatory bowel disease (Genollen et al., 2016; Homef and Torow, 2020; Torow et al., 2023). Epidemiologic and experimental studies suggest that the same is time for type 1 diabetes (T1D) (Hu et al., 2015; Silverman et al., 2017; Clausen et al., 2016; Livanos et al., 2016). While there is a strong genetic contribution to the disease, the rising incidence of T1D over the past few decades provides compelling evidence that environmental factors, such as the microbiome, also impact the risk of developing T1D (Kostic et al., 2015; Stewart et al., 2018; Knip and Siljander, 2016). Despite these findings, effective microbial-based therapies to treat and prevent T1D and other autoimmune disorders remain elusive. Variability in microbiome studies in T1D ( Yurko vestkiy et al., 2013; Kriegl et al., 2011; King and Savertnick, 2011; Alam et al., 2011) suggests that not only do beneficial and

[0012] 1

[0013] 4924-6583-6675, V. 1harmful bacteria species need to be identified, but how, when, and where these microbes interact with the developing immune system also needs to be considered.

[0014] The timing of microbial interactions with the developing immune system profoundly influences long-term immune health (Genollen et al., 2016; Hornef and Torow, 2020; Dhariwala and Scharschmidt, 2021). At birth, mammals are rapidly colonized with maternal and environmental microbes (Penders et al., 2006). In response to the dietary transition from a milk-based to solid food diet, the microbiome dramatically shifts in composition, diversity, and function during the first few years of life, after which it matures into an adult microbiome(Yatsunenko et al., 2012). In mice, this developmental process is more rapid, with mice initiating solid food around days 12-14, weaning from milk by day 21, and developing a microbiome that resembles that of an adult by the end of the 4thweek of life (van Best et al., 2020; Lubin et al., 2023; Al Nabhani et al., 2019). These dramatic changes in the microbiome during weaning correspond with the remarkable development of key components of the immune system that regulate risk for autoimmunity, including the development of peripheral regulatory T cells (pTregs) (Sefik et al. 2015; Ohnmacht et al., 2015), a transition from maternal IgA to endogenously produced IgA (Rogier et al., 2014; Kramer and Cebra, 1995), and an induction of antigen-specific tolerance to commensal microbes and food antigens (Knoop et al., 2017; 2020). This period of microbiota and immune development presents a unique opportunity for pivotal host-microbe interactions to educate the immune system to restrain or induce allergic (Olszak et al., 2012; Cahenzli et al., 2013), inflammatory (Al Nabhani et al., 2019) and potentially autoimmune diseases. Seminal studies by Kasper, Blumberg, and others demonstrated that early-life colonization is required to prevent allergic disease (Olszak et al., 2012; Cahenzli et al., 2013). Eberl, Hornef, and others then discovered that the early-life transition from milk to solid food, which coincides with dramatic microbiome and immune development, is crucial for maintaining long-term homeostasis (Hornef and Torow, 2020; Lubin et al., 2023; Al Nabhani and Eberl, 2020). Altogether, the timing of specific microbial exposures is critical for educating a healthy immune system and may be critical for developing therapeutic approaches to prevent autoimmunity, but additional work is required to establish the parameters for such an effect.

[0015] 2

[0016] 4924-6583-6675, V. 1SUMMARY

[0017] Thus, in accordance with the present disclosure, there is provided a method of immune modulation in a subject comprising orally administering to said subject a composition comprising Clostridium intestinale, Anaerostipes sp., and Parabacteroides distasonis. The modulation may result in reducing the risk of developing autoimmunity or the degree or extent of autoimmunity in said subject. The method may further comprise administering to said subject Lactobacillus johnsonii, Ligilaclobacillus inurinus, Mammaliicoccus sciuri, Kosakonia cowanii, Enterococcus faecalis, and Staphylococcus xylosus. The method may further comprise assessing one or more of (a) induced PD-1 expression, (b) induced IL- 10 production, (c) induced CTLA-4 expression, and / or (c) induced peripheral regulatory T cells, such as CD4 and CD8 T cells prior to and / or after administering.

[0018] The subject may be less six months old or less, 3 months or less, 2 months old or less, 6 weeks old or less, 1 month old or less, or 4 weeks old or less. The subject may be a human subject, a murine subject, or a mammalian subject. The composition may be administered more than once, such as two, three, four, five, six, seven, eight, nine, ten, twelve, fifteen or twenty times. The composition maybe administered daily, every other day, every third day, every four days, even week, every other week, or monthly. The subject may have a genetic, environmental or therapy-related predisposition to developing autoimmunity.

[0019] The method may reduce or diminish risk of autoimmunity relates to celiac disease, diabetes mellitus type 1, Henoch-Schbnlein purpura, systemic lupus erythematosus, Sjogren syndrome, eosinophilic granulomatosis with polyangiitis, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, Addison's disease, rheumatoid arthritis, ankylosing spondylitis, polymyositis, dermatomyositis, multiple sclerosis, inflammatory bowel disease (e.g. Crohn’s disease, ulcerative colitis), or checkpoint inhibitor induced autoimmunity. The method may result in one or more of (a) induced PD-1 expression, (b) induced IL- 10 production, (c) induced CTLA-4 expression, and / or (c) induced peripheral regulatory T cells, such as CD4 and CD8 T cells.

[0020] In particular, the method may result in a reduced severity of type 1 diabetes in the subject, such as a human subject. The subject or human subject may be further treated with insulin. The subject or human subject may exhibit one or more of improved glucose regulation, reduced A1C scores, and / or improved lipid profile.

[0021] Also provided is method of treating a subject with Autism Spectrum Disorder comprising orally administering to said subject a composition comprising Clostridium

[0022] 3

[0023] 4924-6583-6675, V. 1intestinale, Anaerostipes sp., and Parabacteroides distasonis. The method may further comprise administering to said subject Lactobacillus johnsonii, Ligilactobacillus murinus, Mammaliicoccus sciuri, Kosakonia cowanii, Enterococcus faecalis, and Staphylococcus xylosus. The administration may result in improvement of one or more of challenges with social communication / interaction (e.g., poor eye contact, difficulty with back-and-forth conversation), restricted, repetitive patterns of behavior or interests (e.g., hand-flapping, intense focus on specific topics, strict routines), sensory sensitivities (e.g., to light, sound, touch), anxiety, unusual eating patterns, and / or unusual sleeping patterns

[0024] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number.

[0025] It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0026] 4

[0027] 4924-6583-6675, V. 1BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0029] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0030] FIGS. 1A-D. PedsCom colonization prevents diabetes in NOD mice. (FIG. 1A) Schematic of gnotobiotic mice raised in individual gnotobiotic isolators: germfree, Pediatric Community (PedsCom), Complex Mature Community (CMCom). (FIG. IB) Relative abundance of PedsCom members in the small intestines, ceca, and large intestines of adult PedsCom-colonized NOD females. Ml, M2, M3, and M4 denote individual mice. (FIG. 1C) Diabetes incidence in NOD female mice born to PedsCom dams (n=19), germfree dams (n=20), CMCom dams (n=19), or germfree female mice colonized with PedsCom at 6 weeks of age (n=8). (FIG. ID) Composition of PedsCom members in fecal samples from PedsCom NOD mice colonized at birth and germfree NOD mice colonized with PedsCom at 6 weeks of age. Groups compared by the Logrank test, * p< 0.05.

[0031] FIGS. 2A-F. PedsCom induces pTregs in the gut and restrains IFNy in the pancreas. (FIG. 2A) Representative flow cytometry plots of cecal CD4+FOXP3+RORy+Hclios" pTregs. Cells were gated on CD45+TCRb+CD4+FOXP3+cells. (FIG. 2B) Percentages of pTregs of total FOXP3+Tregsin the ceca and PLNs of germfree, PedsCom, and CMCom mice. (FIG. 2C) Percentages of IL-10-producing Tregsin the ceca and PLNs of PedsCom and CMCom mice. (FIG. 2D) Percentages of IL- 10 producing CD4+FOXP3‘ T cells in the ceca and PLNs of PedsCom and CMCom mice. (FIG. 2E) Representative flow cytometry of IFNg-producing CD4+T cells. (FIG. 2F) Percent of IFNg-producing CD4+T cells in the pancreatic islets of PedsCom and CMCom mice. Data in panels A and B are representative of 6 experiments. Groups are compared with the Kruskal-Wallis test. n=4 samples per tissue per gnotobiotic community, ages 5-11 weeks old. GF=germfree, PC=PedsCom, CM=CMCom. Data in panels C-F are representative of 4 experiments. Groups are compared with the

[0032] 5

[0033] 4924-6583-6675, V. 1Welch’s t-test, n=3 tissues per gnotobiotic community, ages 10-12 weeks. *p<0.05, **p<0.01, ****p<0.0001.

[0034] FIGS. 3A-H. Specific PedsCom members induce pTregs or systemic antibody responses. (FIG. 3A) Schematic of PedsCom monocolonization experiments. Three- week-old germfree NOD mice were colonized with individual PedsCom species or with all nine PedsCom (PC) species and analyzed two weeks later. (FIG. 3B) Representative flow cytometry plots of cecal CD4+FOXP3+RORy+I lelios’ pTregs from germfree and monocolonized NOD mice. (FIG. 3C) Percentages of pTregsof total FOXP3+Tregsin the ceca, large intestines, and mesenteric lymph nodes (MLNs) of monocolonized NOD mice. Groups were compared by ANOVA. N=3 mice per PedsCom microbe. (FIG. 3D) Sera from germfree, PedsCom, and RAG2 deficient mice were incubated with pure cultures of individual species from PedsCom. Histogram of microbial flow cytometry of systemic IgGl and IgG2b binding to M. sciuri. (FIGS. 3E-F) Frequency of each PedsCom species bound by IgGl (FIG. 3E) and IgG2b (FIG. 3F) from scrum of germfree or PedsCom mice. L. murinus was excluded due to high levels of nonspecific binding of secondary antibody. (FIG. 3G) Heat map depicting the mean pTregs(CD4+FOXP3+RORy+Helios-) percentages of total FOXP3+Tregsin the large intestines of individual PedsCom monocolonized mice and the mean binding of systemic antibodies to individual PedsCom species. (FIG. 3H) Linear regression of pTregfrequency and IgGl binding to individual PedsCom species. Groups compared by the two-way ANOVA test, n=5 PedsCom mice and n=4 germfree mice, **p<0.01, ****p<0.0001. GF=germfree, PC=PedsCom.

[0035] FIGS. 4A-K. P. distasonis, Anaerostipes sp., and / or C. intestinale are required for the protection from T1D mediated by PedsCom microbes. (FIG. 4A) Comparison of PedsCom and PedsCom-6 community (PedsCom without P. distasonis, C. intestinale, and Anaerostipes sp.)- (FIG. 4B) Diabetes incidence in NOD female mice born to PedsCom dams (n=19), CMCom dams (n=19), or PedsCom-6 dams (n=12). Groups were compared by the Log-rank test. *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. (FIG. 4C) Percentage of FOXP3+Tregsof total CD4+T cells in the pancreatic lymph nodes (PLNs) and spleens of PedsCom and PedsCom-6 mice. (FIG.

[0036] 4D) Percentage of RORy+pTregsof total FOXP3+Trcgsin the ceca and spleens of PedsCom and PedsCom-6 mice. (FIG. 4E) Percentage of MHCII+antigen-presenting cells of total CD1 lc+CD45+cells in the PLNs and spleens of PedsCom and PedsCom- 6 mice. (FIG. 4F) MHCII median fluorescence intensity (MFI) in 6

[0037] 4924-6583-6675, V. 1MHCILCDI lc+CD45+cells in the PLNs and spleens of PedsCom and PedsCom-6 mice. (FIG. 4G) Representative flow cytometry plots of splenic CD44hlFOXP3 CD4+PD-1+T cells from PedsCom and PedsCom-6 NOD mice. Mice were 6 weeks of age. (FIG.

[0038] 4H) Percent PD-1+of effector CD44hiFOXP3 CD4+T cells in the PLNs and spleens of PedsCom and PedsCom-6 mice. Mice were 6 weeks of age. (FIG. 41) Percent PD-1+of effector CD44hlCD8+T cells in the PLNs, spleens, and MLNs of PedsCom and PedsCom-6 mice. (FIG. 4J) Schematic of PedsCom & PedsCom-6 anti-PD-1 experiments. Mice were 6 weeks of age. (FIG. 4K) Diabetes-free survival of 6-10- week-old female NOD mice following treatment with anti-PD-1 antibody or IgG Isotype control: PedsCom + anti-PD-1 (red), PedsCom-6 + anti-PD-1 (black), and PedsCom + Isotype control (yellow).

[0039] FIGS. 5A-B. Specific PedsCom members translocate during early life. (FIG.

[0040] 5A) Live bacteria recovered from homogenates of mesenteric lymph nodes (MLNs), spleens, and livers of prc-wcaning (2-wcck-old, n=8), weaning (3-wcck-old, n=10), and adult mice (6-week-old, n=3). Groups compared with the Kruskal-Wallis test. Representative of two independent experiments. (FIG. 5B) Bacterial genomes detected in MLNs from PedsCom and germfree NOD mice by qPCR. All nine species were analyzed. The four taxa above the limit of detection and with significant differences between GF and PC samples are shown. PedsCom mice (n=ll) and germfree mice (n=3). Groups compared with the Welch’s t-test. *p<0.05, **p<0.01.

[0041] FIGS.6A-B. Microbiome of the Complex Mature Community (CMCom). (FIG.

[0042] 6A) Representative family level taxonomy of fecal microbiota of NOD mice colonized with the CMCom community. CM1, CM2, CM3, CM4 denote individual mice. (FIG.

[0043] 6B) Mean read coverage of PedsCom taxa in fecal microbiome of 6-week-old adult CMCom NOD mice (n=4). B. SPF NOD 2weeks, SPF NOD adults, CMCom FIG.7. Schematic of PedsCom post- weaning colonization.

[0044] FIGS.8A-C. PedsCom induces IL-10-producing T cells. (FIG. 8A) Percentages of pTregs (CD4+FOXP3+RORg+Helios ) of total FOXP3+Tregs in the large intestines, mesenteric lymph nodes (MLNs), and spleens of germfree, PedsCom, and CMCom NOD mice. N=4 samples per tissue per gnotobiotic community, ages 5-11 weeks old. Groups compared with the Kruskal- Wallis test. (FIG. 8B) Representative flow cytometry of IL-10 producing CD4+T cells, CD4+FOXP3+Tregsand RORy+IL-10 producing Tregs. (FIG. 8C) Percentages of IL- 10 producing CD4+T cells in the ceca of germfree, PedsCom and CMCom mice. Percentages of IL- 10 producing Tregs in the ceca 7

[0045] 4924-6583-6675, V. 1of germfree, PedsCom and CMCom mice. Percentages of RORy+Tregs in IL- 10 producing Tregs in the ceca of germfree, PedsCom and CMCom mice. n=3 tissues per gnotobiotic community, ages 10-12 weeks. Groups compared by the Kruskal-Wallis test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. GF=germfree, PC=PedsCom, CM=CMCom.

[0046] FIG. 9. Similar levels of insulitis in PedsCom and CMCom NOD mice.

[0047] Composite insulitis score from NOD female mice at 10-12 weeks of age. n>7 tissues per gnotobiotic community. Groups compared with the Mann-Whitney-Wilcoxon test.

[0048] FIG. 10. Model for microbial-induced protection from autoimmune diabetes mediated by commensal induced IL-10-producing-Tregs.

[0049] FIGS. 11A-B. Specific PedsCom species does not induce systemic IgG3 antibodies. (FIG. 11 A) Sera from germfree, PedsCom, and RAG2 deficient mice were incubated with pure cultures of individual species from PedsCom. Histogram of microbial flow cytometry of systemic, IgG3 binding to M. sciuri. (FIG. 1 IB) Frequency of each PedsCom species bound by IgG3 from serum of gemifree or PedsCom mice. Groups compared by the two-way ANOVA test, n=5 PedsCom mice and n=4 germfree mice, GF=germfree, PC=PedsCom.

[0050] FIG. 12. Microbiome of PedsCom-6 (P6) community. Relative abundance of PedsCom members in feces of P6 and PC (PedsCom) NOD mice.

[0051] FIG. 13. Increased translocation at the MLN during weaning is independent of IgA. Comparison of live bacteria recovered from homogenates of mesenteric lymph nodes (MLNs) of weaning (Day 18-22) and post-weaning (Day 28-35) of IgA' / _(red) and (G) IgA+ / _mice (black). Groups compared with the Mann- Whitney -Wilcoxon test. IgA_ / _weaning (n = 7), post-weaning (n= 11); IgA+ / _peri- weaning (n = 7), post- weaning (n = 3).

[0052] FIGS. 14A-B. PedsCom restrains sialitis in NOD female mice. (FIG. 14A) Representative haematoxylin- and eosin-stained salivary gland sections from female NOD mice with different microbiomes. GF = germ free; PC = PedsCom; CMCom (complete standard microbiome). (FIG. 14B) Quantification of sialitis in 12-to-15 week-old female mice (n = 5 per microbiota group). Each data point represents an individual mouse and lines represent medians. P-value was determined by Mann- Whitney U-test.

[0053] FIGS. 15A-B. PedsCom mice display increased sociability and anxiety. (FIG.

[0054] 15 A) Sociability: The three-chamber sociability test is one of the standard assays for 8

[0055] 4924-6583-6675, V. 1measuring social interaction in rodents. After placing an age and gender matched social target into one side of the chamber under a wire cup and placing a toy object under a cup in the opposite chamber, a subject mouse is allowed to freely explore for 10 minutes. The time spent interacting with the social target is used to calculate sociability. (FIG.

[0056] 15B) Anxiety / open field test: Mice naturally prefer to stay near the comers of an open field, but they also have a strong drive to explore new environments. When less anxious, they enter and explore the center zone. However, when mice are anxious, their exploratory behavior decreases, and they tend to remain in the comers (decreased center time).

[0057] 4924-6583-6675, V. 1DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0058] As discussed above, the early-life gut microbiome educates the developing immune system and has long-lasting impacts on the immune function. How early-life microbiota contributes to autoimmune disease and whether manipulating it can prove therapeutically beneficial remains largely unexplored. To address these questions, the inventors leveraged the early-life microbiome from mice that are genetically protected from type 1 diabetes (T1D) to develop a simple consortium of nine commensal bacteria (PedsCom). They determined that PedsCom colonization robustly prevents type 1 diabetes in NOD mice by enhancing IL- 10 producing T cells in the pancreatic lymph nodes, upregulating inhibitory receptor effector cells and suppressing IFNy-producing CD4 T cells in the pancreatic Islets. Remarkably, the inventors found that this microbial protection from T1D is completely dependent upon early-life colonization. During this critical development window of early-life colonization, the immunomodulatory microbes Parabacteroides distasonis and a novel Anaerostipes species unexpectedly translocate from the gut to the mesenteric lymph nodes and induce the tolerogenic responses required for T1D protection. These findings highlight how the timing and localization of microbial interactions during a pivotal stage of immune development contribute to protection from T1D. In addition, data also show that the microbes can reduce abnormal behaviors in a model of Autism Spectrum Disorder (ASD). Altogether, these findings suggest an opportunity to develop microbial therapies for human infants to prevent autoimmune diseases.

[0059] These and other aspects of the disclosure are described in detail below.

[0060] I. PedsCom

[0061] A. General Background

[0062] The present inventors have previously described a microbiome preparation derived from 14-day-old Eal6 / NOD mice (Lubin et al., 2023). This preparation comprises nine different microorganisms, including Clostridium intestinale, Anaerostipes sp., Parabacteroides distasonis, Lactobacillus johnsonii, Ligilactobacillus murinus, Mammaliicoccus sciuri, Kosakonia cowanii, Enterococcus faecalis, and Staphylococcus xylosus. Of particular interest is a three-microbe subset including P. distasonis, Anaerostipes sp., and / or C. intestinale, which are required for protection against type 1 diabetes (T1D) conferred by PedsCom.

[0063] 10

[0064] 4924-6583-6675, V. 1Clostridium intestinale, Anaerostipes sp., and Parabacteroides distasonis may be grown anaerobically at 37°C in brain heart infusion (BHI) media (Oxoid, UK) supplemented with hemin (5 mg / L) and vitamin K (0.5 mg / L) (BD, Franklin Lakes, NJ). Lactobacillus johnsonii and Ligilactobacillus murinus may be grown anaerobically at 37°C in de Man Rogosa Sharpe (MRS) media (Sigma, MO, USA). Mammaliicoccus sciuri, Kosakonia cowanii, Enterococcus faecalis, and Staphylococcus xylosus may be grown in ambient air al 37°C in BHI media.

[0065] The preparation may be produced starting from liquid cultures that are centrifuged and resuspended in sterile reduced buffer, such as phosphate-buffered saline containing 0.1% L-cysteine (rPBS). The optical density (OD) of cultures can be determined using a spectrophotometer. Bacterial concentration may be adjusted and aliquoted to 10scolonyforming units (CFU) for use in monocolonization.

[0066] The component bacteria in the PedsCom consortium used in the Examples below has been completely sequenced and their sequences previously disclosed. The details arc shown in Table 2, below.

[0067] B. Handling and Storage

[0068] PedsCom is stored as glycerol stocks at -80 °C either as individual species or the entire PedsCom consortia. It can be formulated for administration by resuspending in a pooled sample of 1 x 109colony forming units (CFU) of each isolate in reduced PBS and administer 100 pl to each mouse. One can grow and lyophilize the PedsCom microbes to create solid formulations, such as pills or tablets.

[0069] A model protocol for preparing glycerol stocks is as follows:

[0070] • Bacterial culture

[0071] • Set up 1-5 ml cultures of bacteria to be stocked.

[0072] • Reseed cultures in 5 ml of fresh media grow bacteria log phase for storage.

[0073] ■ Actively growing (log phase) are more viable and survive the freezing process better.

[0074] • Glycerol stock preparation

[0075] • Label sterile cryotubcs with: 1. Date 2. Stock# 3. Species and strain name 4. Any antibiotic resistances 5. (If applicable) Plasmid / vector the strain contains:

[0076] ■ For new bacteria make at least 2 stocks, preferably 4.

[0077] 11

[0078] 4924-6583-6675, V. 1Add 0.75 ml from the log phase cultures and 0.75 ml of sterile 50% glycerol- BHI to the cryotubes.

[0079] • Mix well with vortex before storage to ensure even distribution of glycerol. • Freeze at -80C only, stocks will die at -20 °C.

[0080] • The following day restreak a sample to ensure viability of frozen stock ■ Use a 1000 pl pipette tip to scrap a small chunk of frozen stock on to the appropriate agar plate, perform quadrant streak and incubate.

[0081] • Notes

[0082] • Final glycerol concentration in frozen stock will be 25%.

[0083] • Do not let frozen stocks thaw when using. Keep stocks on ice when using.

[0084] Dry ice is preferable, stocks will start to thaw after a while even on ice. Use only dry ice in anerobic chamber. Make sure containers containing dry ice and stocks in pass box is sealed (not too tight) to prevent sublimation during vacuuming step.

[0085] • Multiple usage will also affect viability. Use one duplicate stock at a time.

[0086] • Solutions - 50% glycerol-BHI (50 ml)

[0087] • BHI provides nutrients to yield better survivabilily, but is not absolutely necessary

[0088] ■ Prepare 25 ml of BHI in a beaker according to manufacturer’s instructions.

[0089] ■ In a 100 ml screw cap bottle, measure out 25 ml of glycerol

[0090] • Glycerol is very viscous. I find it easier to measure out the 25 ml by mass instead of using a grad cylinder.

[0091] • Density of glycerol: 1.26 g / ml - for 25 ml, weigh out 31 ,5g on scale in a tared bottle.

[0092] ■ Pour BHI into glycerol bottle and stir to mix

[0093] ■ Autoclave for 15-20 min at 121 °C.

[0094] IL Autoimmunity

[0095] Autoimmunity is the system of immune responses of an organism against its own healthy cells, tissues and other normal body constituents. Any disease resulting from this type of immune response is termed an “autoimmune disease.” Autoimmunity results from the presence of antibodies or T cells that react with self-protein and is present in all individuals,

[0096] 12

[0097] 4924-6583-6675, V. 1even in normal health state. It causes autoimmune diseases if self-reactivity can lead to tissue damage.

[0098] Prominent examples include celiac disease, diabetes mellitus type 1, Henoch-Schonlein purpura, systemic lupus erythematosus, Sjogren syndrome, eosinophilic granulomatosis with polyangiitis, Hashimoto's thyroiditis, Graves’ disease, idiopathic thrombocytopenic purpura, Addison's disease, rheumatoid arthritis, ankylosing spondylitis, polymyositis, dermatomyositis, and multiple sclerosis. Autoimmune diseases are very often treated with steroids.

[0099] Autoimmune diseases can be broadly divided into systemic and organ-specific or localized autoimmune disorders, depending on the principal clinico-pathologic features of each disease.

[0100] Systemic autoimmune diseases include coeliac disease, lupus erythematosus, Sjogren syndrome, scleroderma, rheumatoid arthritis, cryoglobulinemic vasculitis, and dermatomyositis. These conditions tend to be associated with autoantibodies to antigens which arc not tissue specific. Thus, although polymyositis is more or less tissue specific in presentation, it may be included in this group because the autoantigens are often ubiquitous t-RNA synthetases.

[0101] Local syndromes which affect a specific organ or tissue include diabetes mellitus type 1, Hashimoto's thyroiditis, Addison's disease, pernicious anaemia, pemphigus vulgaris, vitiligo, autoimmune haemolytic anaemia, idiopathic thrombocytopenic purpura, multiple sclerosis, myasthenia gravis, autoimmune encephalitis, and gluten ataxia.

[0102] Using the traditional "organ specific" and "non-organ specific" classification scheme, many diseases have been lumped together under the autoimmune disease umbrella. However, many chronic inflammatory human disorders lack the telltale associations of B and T cell driven immunopathology. More recently, it has been firmly established that tissue "inflammation against self" does not necessarily rely on abnormal T and B cell responses. This has led to the recent proposal that the spectrum of autoimmunity should be viewed along an "immunological disease continuum", with classical autoimmune diseases at one extreme and diseases driven by the innate immune system at the other extreme. Within this scheme, the full spectrum of autoimmunity can be included. Many common human autoimmune diseases can be seen to have a substantial innate immune mediated immunopathology using this new scheme. This new classification scheme has implications for understanding disease mechanisms and for therapy development.

[0103] Treatments for autoimmune disease have traditionally been immunosuppressive, antiinflammatory, or palliative. Managing inflammation is critical in autoimmune diseases. Non- 13

[0104] 4924-6583-6675, V. 1immunological therapies, such as hormone replacement in Hashimoto's thyroiditis or Type 1 diabetes mellitus, treat outcomes of the autoaggressive response, and thus these are palliative treatments. Dietary manipulation limits the severity of celiac disease. Steroidal or NSAID treatment limits inflammatory symptoms of many diseases. IVIG is used for CIDP and GBS. Specific immunomodulatory therapies, such as the TNFa antagonists (e.g. etanercept), the B cell depleting agent rituximab, the anti-IL-6 receptor tocilizumab and the costimulation blocker abatacept have been shown to be useful in treating RA. Some of these immunotherapies may be associated with increased risk of adverse effects, such as susceptibility to infection. Helminthic therapy is an experimental approach that involves inoculation of the patient with specific parasitic intestinal nematodes (helminths). There are currently two closely related treatments available, inoculation with either Necator americanus, commonly known as hookworms, or Trichuris Suis Ova, commonly known as Pig Whipworm Eggs. T-cell vaccination is also being explored as a possible future therapy for autoimmune disorders.

[0105] Here, the inventors propose the use of a PcdsCom bacterial consortium for oral administration to a subject to effect immune modulation. The method of immune modulation in a subject comprises use of a composition comprising Clostridium intestinale, Anaerostipes sp., and Parabacteroides distasonis, optionally using up to six additional bacterial strains. The modulation provided reduces the risk of developing autoimmunity or degree of autoimmunity in said subject in a variety of different contexts.

[0106] An under-appreciated truth in immunology is that everyone has auto-reactive T and B cell. As such, multiple inhibitory and regulatory pathways are essential to prevent the development of clinical autoimmune disease. Inhibitory receptors, such as PD-1, and its ligand PD-L1, are crucial for preventing multiple autoimmune diseases. Indeed, autoimmunity is one of the most common adverse events triggered by anti-PD-1 therapy, and human patients with loss of functional mutations in the PD-1 gene develop early-onset multi-system autoimmune disease. As such, the ability to modulate the level of PD-1 on activated autoreactive T cells via microbiome modulation may be an effective therapeutic strategy.

[0107] NOD mice are a valuable model of T1D and other autoimmune disease including sjogren’s and autoimmune thyroiditis (Anderson & Bluestone, 2005). The inventors can extrapolate from NOD mice to other autoimmune diseases given that PD-1 inhibition of activated T cells, which occurs after interaction with its ligand PD-L1, critically regulates autoimmune diabetes (Collier et al., 2023) and sialitis (Sjogren’s model) in NOD mice (Zhou et al., 2016), and PD-1 inhibition is critical to prevent other autoimmune diseases. Indeed, autoimmunity is one of the most common adverse events triggered by anti-PD- 1 or anti-PD- 14

[0108] 4924-6583-6675, V. 1LI therapy (Martins et al., 2019), and human patients with loss of functional mutations in the PD-1 or PD-L1 gene develop early-onset multi-system autoimmune disease (Johnson et al., 2024; Ogishi et al., 2021). As such, the ability to modulate the level of PD-1 on activated autoreactive T cells via microbiome modulation should constitute an effective therapeutic strategy across a wide range of autoimmune disfunction.

[0109] III. Treatment of Disease

[0110] The present inventors envision treating a wide variety of disorders and diseases with the present microbiome described herein. The following are mere examples of such disorders and diseases.

[0111] A. Type 1 Diabetes

[0112] In a particular embodiment, the present disclosure addresses prevention or mitigation of Type 1 diabetes (a.k.a. T1D), formerly known as juvenile diabetes. T1D is an autoimmune disease that occurs when pancreatic cells (beta cells) are destroyed by the body's immune system. In healthy subjects, beta cells produce insulin. Insulin is a hormone required by the body to store and convert blood sugar into energy. T1D results in high blood sugar levels in the body prior to treatment. Common symptoms include frequent urination, increased thirst, increased hunger, weight loss, and other complications. Additional symptoms may include blurry vision, tiredness, and slow wound healing (owing to impaired blood flow). While some cases take longer, symptoms usually appear within weeks or a few months. Type 1 diabetes can typically be distinguished from type 2 diabetes by testing for the presence of autoantibodies and / or declining levels / absence of C-peptide.

[0113] The cause of type 1 diabetes is not completely understood, but it is believed to involve a combination of genetic and environmental factors. The underlying mechanism involves an autoimmune destruction of the insulin-producing beta cells in the pancreas. Diabetes is diagnosed by testing the level of sugar or glycated hemoglobin (HbAlC) in the blood.

[0114] Currently, there is no known way to prevent type 1 diabetes. Treatment with insulin is required for survival. Insulin therapy is usually given by injection just under the skin but can also be delivered by an insulin pump. A diabetic diet, exercise, and lifestyle modifications are considered cornerstones of management. If left untreated, diabetes can cause many complications. Complications of relatively rapid onset include diabetic ketoacidosis and nonketotic hyperosmolar coma. Long-term complications include heart disease, stroke, kidney

[0115] 15

[0116] 4924-6583-6675, V. 1failure, foot ulcers, and damage to the eyes. Furthermore, since insulin lowers blood sugar levels, complications may arise from low blood sugar if more insulin is taken than necessary.

[0117] Type 1 diabetes makes up an estimated 5-10% of all diabetes cases. The number of people affected globally is unknown, although it is estimated that about 80,000 children develop the disease each year. Within the United States the number of people affected is estimated to be one to three million. Rates of disease vary widely, with approximately one new case per 100,000 per year in East Asia and Latin America and around 30 new cases per 100,000 per year in Scandinavia and Kuwait. It typically begins in children and young adults but can begin at any age.

[0118] B. Sjogren’s Disease

[0119] Sjogren’s disease (also called Sjogren's syndrome) is a chronic systemic autoimmune disorder characterized by immune-mediated destruction and dysfunction of the exocrine glands, most prominently the salivary and lacrimal glands, leading to sicca symptoms of dry mouth (xerostomia) and dry eyes (keratoconjunctivitis sicca). It can occur as a primary disease or in association with other autoimmune conditions such as rheumatoid arthritis or systemic lupus erythematosus. Pathogenesis involves lymphocytic infiltration of target tissues, aberrant B-cell activation, autoantibody production (classically anti-SSA / Ro and anti-SSB / La), and type I interferon-driven immune pathways. Beyond glandular involvement, Sjogren’s disease is a systemic condition with potential extraglandular manifestations affecting the musculoskeletal, pulmonary, renal, neurologic, and vascular systems, and it carries an increased risk of B-cell non-Hodgkin lymphoma. Diagnosis integrates clinical features, serologic markers, objective tests of glandular function, and sometimes minor salivary gland biopsy, while management focuses on symptomatic relief of sicca symptoms and immunomodulatory therapy for systemic disease.

[0120] C. Behavioral Abnormalities Consistent with Autism Spectrum Disorder Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by persistent differences in social communication and social interaction, along with restricted, repetitive patterns of behavior, interests, or activities, with symptoms typically emerging in early childhood and varying widely in severity and presentation. The disorder reflects a spectrum of phenotypes shaped by complex interactions among genetic susceptibility, neurobiological development, and environmental influences, leading to altered brain connectivity and information processing. Individuals with ASD may also exhibit sensory

[0121] 16

[0122] 4924-6583-6675, V. 1processing differences, language delays or atypical language use, and co-occurring conditions such as intellectual disability, attention-deficit / hyperactivity disorder, anxiety, epilepsy, or gastrointestinal symptoms. Diagnosis is clinical, based on standardized behavioral assessments and developmental history, and early identification is important because evidence-based behavioral, educational, and supportive interventions can significantly improve functional outcomes and quality of life.

[0123] IV. Formulation and Administration

[0124] The present disclosure provides bacterial compositions as described in Section I, above. Such compositions comprise a prophylactically or therapeutically effective amount of the bacterial consortium, and a optionally pharmaceutically acceptable carrier. In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.

[0125] The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which is primarily oral, but may be intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation.

[0126] 17

[0127] 4924-6583-6675, V. 1Pharmaceutically acceptable salts include the acid salts and those which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

[0128] Generally, ingredients of the compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. The compositions may be provided in a concentrated form, along with a diluent and mixed prior to administration.

[0129] Probiotics generally have higher requirements on the environment and poorer tolerance to the extreme conditions of long-term storage at normal temperature. Nonetheless, many probiotics arc supplied in the form of frcczc-dricd powder in the market. Thus, there is a need to avoid damage to cells in the extremes of freeze-drying process, and use of freeze-drying protective agents in the freeze-drying process and greatly improves the survival rate of the cells. At present, freeze-drying protective agents commonly used mainly contain sugars like sucrose and trehalose, sugar alcohols like sorbitol and mannitol, skimmed milk powder, maltodextrin, protein, glycerol, betaine, adonitol, and certain types of gums like alginate and xanthan gum.

[0130] As indicated herein, a product of the present disclosure can take the form of a food product including, but is not limited to, a health bar, health drink, yogurt, dahi, or sachet or an enterically coated tablet, capsule, powder, soft gel, gelcap, or liquid. In addition to containing at least one, at least two or at least three probiotic components, symbiotic product of the present disclosure can further contain various fillers or additives.

[0131] Optional additives of the present composition include, without limitation, pharmaceutical excipients such as magnesium stearate, talc, starch, sugars, fats, antioxidants, amino acids, proteins, nucleic acids, electrolytes, vitamins, derivatives thereof or combinations thereof. In one embodiment, an additive of the product is carob flour, for example, locust bean gum. In another embodiment, an additive is a mushroom extract from Agaricus bisporus. In particular embodiments, a gel cap contains fillers such as magnesium stearate, talc and starch.

[0132] Further, to increase the palatability of a food product containing a prebiotic and probiotic, it may be desirable to add flavors, sweetening agents, binders or bulking agents.

[0133] Flavors which can optionally be added to the present compositions are those well-known in the pharmaceutical arts. Examples include, but are not limited to, synthetic flavor 18

[0134] 4924-6583-6675, V. 1oils, and / or oils from plants leaves, flowers, fruits and so forth, and combinations thereof are useful. Examples of flavor oils include, but are not limited to, spearmint oil, peppermint oil, cinnamon oil, and oil of wintergreen (methylsalicylate). Also useful are artificial, natural or synthetic fruit flavors such as citrus oils including lemon, orange, grape, lime, and grapefruit, and fruit essences including apple, strawberry, cherry, pineapple and so forth.

[0135] Sweetening agents can be selected from a wide range of materials such as water-soluble sweetening agents, water-soluble artificial sweeteners, and dipeptide-based sweeteners, including salts thereof and mixtures thereof, without limitation.

[0136] Binders can be selected from a wide range of materials such as hydroxypropylmethylcellulose, ethylcellulose, or other suitable cellulose derivatives, povidone, acrylic and methacrylic acid co-polymers, pharmaceutical glaze, gums (e.g., gum tragacanth), milk derivatives (e.g., whey), starches (e.g., com starch) or gelatin, and derivatives, as well as other conventional binders well-known to persons skilled in the art. Examples of bulking substances include, but arc not limited to, sugar, lactose, gelatin, starch, and silicon dioxide.

[0137] When the above-mentioned additives are included in the product of the present disclosure, they are generally less than 15% of the total product weight. In particular embodiments, they are less than 5 to 10% of the total product weight.

[0138] To facilitate targeting of the product of the present disclosure to the gastrointestinal tract, there a number of controlled release formulations that are developed preferably for oral administration. These include, but are not limited to, osmotic pressure-controlled gastrointestinal delivery systems; hydrodynamic pressure-controlled gastrointestinal delivery systems; membrane permeation-controlled gastrointestinal delivery systems, which include microporous membrane permeation-controlled gastrointestinal delivery devices; gastric fluidresistant intestine targeted controlled-release gastrointestinal delivery devices; gel diffusion-controlled gastrointestinal delivery systems; and ion-exchange-controlled gastrointestinal delivery systems, which include cationic and anionic drugs. Additional information regarding controlled release drug delivery systems can be found in Yie W. Chien, Novel Drug Delivery Systems, 1992 (Marcel Dekker, Inc.).

[0139] For example, enteric coatings are applied to tablets to prevent the release of probiotics in the stomach either to reduce the risk of unpleasant side effects or to maintain the stability of the drug which might otherwise be subject to degradation of expose to the gastric environment. Most polymers that are used for this purpose are polyacids that function by virtue or the fact that their solubility in aqueous medium is pH-dependent, and they require conditions with a pH higher than normally encountered in the stomach. One desirable type of oral controlled 19

[0140] 4924-6583-6675, V. 1release structure is enteric coating of a solid or liquid dosage form. Enteric coatings promote the compounds remaining physically incorporated in the dosage form for a specified period when exposed to gastric juice. Yet the enteric coatings are designed to disintegrate in intestinal fluid for ready absorption. Delay of absorption is dependent on the rate of transfer through the gastrointestinal tract, and so the rate of gastric emptying is an important factor. Some investigators have reported that a multiple-unit type dosage form, such as granules, may be superior to a single-unit type.

[0141] Typical enteric coating agents include, but are not limited to, hydroxypropylmethylcellulose phthalate, methacryclic acid-methacrylic acid ester copolymer, polyvinyl acetate-phthalate and cellulose acetate phthalate (Hasegawa, (1985) Chem. Pham. Bull. 33:1615-1619). Various enteric coating materials can be selected on the basis of testing to achieve an enteric-coated dosage form designed ab initio to have a preferable combination of dissolution time, coating thicknesses and diametral crashing strength. (Porter et al. (1970) J. Pharm. Pharmacol. 22:42p). It is contemplated that the enteric coating can be cither food grade or pharmaceutical material which is generally used in the production of various drag or dietary supplements.

[0142] Depending on whether the product is to be consumed by an adult human, child or animal (e.g., companion animal or livestock), it can be produced in various sizes and with various ingredients suitable for the intended recipient. For example, while a gel cap size of 0 or 1 may be suitable for humans, a gel cap size of 2, 3, 4, or 5 may be more suitable for a companion animal.

[0143] Further, because the probiotic and prebiotic components of the present disclosure are generally recognized as safe, they can be consumed one, two or three times daily or more.

[0144] V. Examples

[0145] The following examples are included to demonstrate preferred embodiments. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of embodiments and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.

[0146] 20

[0147] 4924-6583-6675, V. 1Example 1 - Materials and Methods

[0148] Mice. Germfree NOD mice were generated by Caesarian-section delivery and crossfostered to germfree Swiss-Webster mice. Gnotobiotic mice were bred and housed in the Hill Pavilion vivarium at the University of Pennsylvania. Mice were fed the Autoclaved LabDiet 5021 (Cat# 0006540) ad libitum and housed on autoclaved Beta-chip hardwood bedding (Nepco, NY, USA). To generate the PedsCom NOD gnotobiotic line, germfree 6 to 8-week-old NOD mice were gavaged with 109CFU of each of the 9 PedsCom species in 100 mL of reduced PBS (rPBS). To generate the CMCom gnotobiotic line, whole cecal contents of a 6-week-old female SPF Eal6 / NOD mouse were collected anaerobically in 5mL rPBS, the contents settled for 10 mins, and then 100 mL of the supernatant was used as donor microbiota material for oral gavage (Lubin et al., 2023). Gavaged mice were bred in separate PedsCom and CMCom gnotobiotic isolators, then Fl and later generations were used experimentally, to ensure natural vertical transmission of microbiota. Sterility checks were regularly performed on germfree isolators each month. Freshly collected pellets were cultured on brain heart infusion (BHI) (Oxoid, UK), NB 1, and Sabouraud media for 65-70 hours at 37°C under aerobic and anaerobic conditions with positive and negative control samples. Isolator sterility was confirmed externally every 3-4 months by Charles River Laboratories (NJ, USA). PedsCom gnotobiotic line was confirmed by 16sRNA gene metagenomic sequencing every 3-4 months. Unless otherwise indicated, PedsCom and CMCom colonized NOD mice acquired their microbiota via vertical transmission from their dams at birth.

[0149] For monocolonization experiments, NOD mice at 3 weeks of age were orally gavaged with 50 mL of rPBS, 108CFU of individual PedsCom species in 50 mL of rPBS, or 50 mL of cecal contents of 8-week-old female PedsCom NOD mouse that was collected from a 5 mL slurry in rPBS. Gavages were performed using a sterile syringe and a straight 1 -inch-long 22-gauge gavage needle (Cadence Science, #7901). For post-weaning colonization experiments, 6-week-old germfree NOD females were transferred to the PedsCom gnotobiotic isolator and cohoused with female PedsCom mice. All mouse experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Pennsylvania.

[0150] Bacteria. Individual PedsCom species were isolated and cultured from 14-day-old Eal6 / NOD mice, as previously described (Lubin et al., 2023). Clostridium intestinale, Anaerostipes sp., and Parabacteroides distasonis were grown anaerobically at 37°C in brain heart infusion (BHI) media (Oxoid, UK) supplemented with hemin (5 mg / L) and vitamin K (0.5 mg / L) (BD, Franklin Lakes, NJ). Lactobacillus johnsonii and Ligilactobacillus murinus

[0151] 21

[0152] 4924-6583-6675, V. 1were grown anaerobically at 37°C in de Man Rogosa Sharpe (MRS) media (Sigma, MO, USA). Mammaliicoccus sciuri, Kosakonia cowanii, Enterococcus faecalis, and Staphylococcus xylosus were grown in ambient air at 37°C in BHI media. Liquid cultures were centrifuged at 4700 x g at 4°C and resuspended in sterile reduced, phosphate-buffered saline containing 0.1% L-cysteine (rPBS). The optical density (OD) of cultures was determined using a spectrophotometer and the bacteria concentration was adjusted to an O.D. of 0.1 for microbial flow cytometry experiments and 108colony-forming units (CFU) for experiments requiring monocolonization. To introduce the entire PedsCom consortium or a different combination of the PedsCom microbes, mice were gavaged intragastrically with 100 ml of a pooled sample of IxlO9colony forming units (CFU) of each isolate in rPBS to generate the PedsCom gnotobiotic line.

[0153] Diabetes and insulitis. To assess diabetes in gnotobiotic mice, mice were screened for glucosuria starting at 10 weeks of age. Urine was collected and analyzed for glucosuria by dipstick (Diastix; Ames). After two consecutive positive tests for glucosuria, diabetes was confirmed by blood glucose greater than 250 mg / dL. Mice were monitored until 30 weeks of age. To assess insulitis, pancreata were harvested at 10 weeks of age and fixed in 10% formalin. Then pancreata were embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Insulitis was evaluated by a blinded researcher using light microscopy. Islets were scored 0 for no signs of insulitis, 1 for peri-insulitis, and 2 for insulitis. Each mouse was given an average composite score of insulinitis (range 0-2).

[0154] Microbial flow cytometry (mFLOW). Cultured bacteria were centrifuged at 4700 x g for 10 minutes, resuspended in 1 mL PBS, 1% BSA (PBS-BSA), and diluted to an OD600 of 0.1. Bacteria were washed with PBS-BSA, resuspended in blocking buffer (PBS-BSA, 20% normal rat serum), and incubated for 30 minutes at 4°C. The bacteria were then incubated with serum (heat inactivated at 56°C for 30 min, centrifuged at 16000 x g for 5 min at 4°C) diluted 1 :25 in bacterial staining buffer (BSB) (PBS, 1% BSA plus sodium azide) and incubated at 4°C for 1 hr. The bound bacteria were then washed with BSB and incubated with anti-mouse antibodies PE-Cy7-IgGl, clone RMG1-1; BV711-IgG2b, clone R12-3; BV421-IgG3, clone R40-82) diluted 1:25 in BSB for 30 minutes at 4°C, washed and incubated in SytoBC nucleic acid stain (Invitrogen #S34855) diluted 1:500 in Tris-buffered saline (TBS) for 15 minutes at room temperature. Flow cytometry analysis on stained bacteria populations with SytoBC was used to define living bacteria. RAG2 KO sera was used to define gates for Ig-coated bacteria and control for nonspecific binding of secondary antibodies. Notably, L. murinus demonstrated

[0155] 22

[0156] 4924-6583-6675, V. 1nonspecific binding of secondary antibodies and was excluded from further analysis. PedsCom samples were compared to sera from germfree mice to determine if the antibody response was elicited from colonization rather than natural antibodies. Microbial flow cytometry analysis was completed on the LSR Fortessa and data analysis was performed using FlowJo vlO software (BD).

[0157] Reverse transcription quantitative PCR. Fecal pellets or intestinal contents were collected in sterile microcentrifuge tubes and stored at -80°C. Bacterial DNA was isolated from samples using the DNAeasy PowerSoil kit (Qiagen, Germany) using a QIACube (Qiagen, #9001292). PedsCom species abundance was determined by qPCR, using TaqMan Master Mix (Thermo-Fisher Scientific, Waltham, MA) and primers for the DNA-directed RNA polymerase subunit beta (rpoH) gene for each specific PedsCom species. Primer and probe sets were generated using the PrimerQuest Tool (Integrated DNA Technologies, IA, USA) and compared with the Multiple Primer Analyzer tool (Thermo-Fisher Scientific, Waltham, MA) (Lubin et al., 2023). A standard curve using serial dilution of cultured bacteria was used to estimate the genomes of each PedsCom species in a sample and determine the limit of detection of the assay for each PedsCom member.

[0158] 16S rRNA gene inetagenomic sequencing. The PennCHOP microbiome program sequencing core sequenced the V4 variable region of the 16S rRNA gene using the Illumina MiSeq platform as previously described (Caporaso et al., 2012). QIIME2 (ver. 2018.2) was used to analyze the resulting sequencing libraries and deblur was used to cluster and de-noise the results into amplicon sequence variants (Bolyen et al., 2019). The Greengenes 99% rRNA gene reference database (ver. 13.8) was used to classify bacterial taxa. Taxa bar plots were generated with the R package phyloseq (McMurdie and Holmes, 2013).

[0159] Isolation of immune cells from tissues. Mouse spleen, MLN, PLN, cecum, and large intestine tissues were isolated from euthanized mice and placed in cell culture media (CCM: RPMI, 100 units / mL penicillin, 100 pg / mL streptomycin, 10% fetal bovine serum (FBS)). The lymph nodes and spleen were dissociated using the back of the plunger of a 1 mL syringe through a 70 pM filter in 1 mL CCM in a dish. The dish and filter were rinsed with CCM, and the suspension was spun down at 480 x g for 10 minutes in 15 mL conical tubes. The supernatant was discarded, and MLNs and PLNs pellets were resuspended in 200 pL CCM and plated in a 96-well round-bottom plate for staining. Splenocytes were resuspended in 1 mL ACK lysis buffer for 1 minute and then quenched with 10 mL CCM and centrifuged as before. The pellet was then resuspended in 1 mL staining media (RPMI, 4% FBS) and 100 pL of each

[0160] 23

[0161] 4924-6583-6675, V. 1sample was plated for staining. The intestines were cleared of debris by gently flushing with cold PBS using a gavage needle or by cutting open the cecum and shaking in CCM. The cecal patch was carefully excised. The colon was rolled on a paper towel and a scalpel was used to remove mesenchyme. All tissues were then cut open and shaken to remove the remaining intestinal debris. The intestinal tissues were then incubated in intestinal epithelial layer (IEL) digestion media (30 mL RPMI, 0.062% dithiothreitol, 1 pM EDTA, 1.3% FBS) and incubated at 37°C for 15 minutes while stirring at 300 rpm with a magnetic bar to remove IEL cells. The tissues were washed with PBS, placed in RPMI, and diced into small sections (~1 mm in size) using dissection scissors. The tissues were then digested for 40 minutes in 25 mL digestion media (25 mL RPMI, 12.5 mg dispase (Gibco), 37.5 mg collagenase type II (Gibco), 1 mL PBS) at 37°C with stirring at 300 rpm to generate a single cell suspension. The suspension was filtered through 100 |_lM filters and quenched with 25 mL RPMI. The solutions were centrifuged for 10 minutes at 310 x g at 4°C and resuspended in FACS buffer (DPBS, 0.1% BSA, 2 mM EDTA).

[0162] The pancreas was perfused with 0.5 mLof C-solution (HBSS, 0.35g / LNaHCO?, 2.5 % bovine serum albumin (BSA), collagenase P 2 mg / mL (Roche, Mannheim, Germany, #11213857001), then carefully removed. The pancreas was incubated in 10 mL C-solution in a water bath at 37° C for 12 minutes. The pancreas tissue was hand-shaken vigorously for 30 seconds to break up the loose tissue. The pancreas tissue was washed three times with 15 mL cold G-solution (HBSS, 0 .35g / LNaHCO3, 2.5 % BSA) to remove the collagenase by spinning down at 4°C at 220 x g for 5 minutes. The tissue homogenate in 50 mL cold G-solution was then filtered through a size 40 sieve (425 um diameter wire mesh, Bellco Glass Cat # 1985-00040) to remove the remaining undigested tissue, fat, and lymph. The tissue homogenate was then centrifuged at 4°C at 220 x g for 5 minutes and resuspended in Llg / mL islet suspension by mixing 7.5 mL Histopaque 1.119 (Sigma) with 3.5 mL Histopaque 1.077 (Sigma). 7 ml of Histopaquc 1.119 was added under the islet phase using a needle. 12 ml Histopaque 1.077 was overlay ed, and then 12 mL of G solution was overlayed on the Histopaque gradient. The gradient solution was then centrifuged for 25 minutes at 1750 x g with very slow acceleration and no braking (1 / 0) at 4°C. The islet layer was collected from each of the interfaces by aspirating 10 mL at each interface. The islets were then washed with 50mL G-solution and centrifuged at 220 x g for 5 minutes to remove Histopaque. The islets were then incubated with 5 mL of 0.1 mM EDTA RPMI solution for 5 minutes. The islets were then washed again and resuspended in 5 mL of pre-warmed dispase solution (Img / mL in RPMI). The islets were

[0163] 24

[0164] 4924-6583-6675, V. 1incubated for 15 minutes at 37° C to dissociate islets into a single-cell suspension. The samples were then centrifuged at 4°C at 220 x g for 5 minutes and resuspended in FACS buffer in preparation for staining.

[0165] Flow cytometry of immune cells. Staining for surface markers of immune cells was carried out in the dark at 4°C for 15 - 30 minutes using the following antibody panel: BV510-CD45 (clone 30F11; Biolegend, CA, USA), PE-Cy7-TCR0 (clone H57-597; Biolegend), APC-Cy7-CD19 (clone 6D5; Biolegend), AF700-CD8 (clone 53-6.7; Biolegend), FITC-CD4 (clone Rm4-5; Biolegend). Cells were washed twice with staining media and fixed overnight at 4°C using the eBioscience Fixation kit (cat# 005223-56, 00-5123-43). Cells were washed twice in permeabilization buffer (Invitrogen cat# 00-8333-56), then centrifuged at 1340 x g and stained for intracellular proteins for 50 minutes at room temperature using the following panel: APC-Foxp3 (clone FJK-16s; eBioscience, MA, USA), PE-RORy (clone B2D; eBioscience), Pacific Blue-Helios (clone 22F6; Biolegend). Cells were washed twice with permeabilization buffer, resuspended in 200 pF of PBS, and filtered through 50 pM nylon mesh (Genesee Scientific, cat# 57-106) into FACS tubes. To detect intracellular cytokines, cells were re-suspended in 200 pF of cell stimulation cocktail with protein transport inhibitors (Thermo-Fisher Scientific, Cat: 00-4975-03) in CCM for 2 hours at 37°C and 5% CO2. Cells were then washed, resuspended, surface stained, and permeabilized as described above. The samples were then intracellularly stained with IL-10-BV421 (clone JES5-16E3; Biolegend) and IFNy-AF488 (clone XMG1.2; Biolegend) as described above. The cells were washed and then resuspended in 500 pl FACS buffer for analysis. Stained samples were analyzed on the LSRFortessa (BD) or Aurora CyTek and data analysis was performed using FlowJo vlO software (BD).

[0166] Microbial translocation. MLNs, spleen, and a segment of liver were sterilely harvested and placed into autoclaved pre- weighed 1.5-mL Eppendorf tubes. The tissue weights were then recorded. Tissue samples were then processed in an anaerobic chamber (90% N2, 5% CO2, and 5% H2). Samples were homogenized in sterile rPBS. Portions of the tissue homogenates were then inoculated onto Yeast Casitone Fatty Acids with Carbohydrate (YCFAC) plates, then incubated anaerobically at 37°C for 48 hours. After incubation, colonyforming units (CFUs) were counted and normalized to the weight of tissue (CFU per gram). A pseudo-count of +1 was used for samples with no growth to visualize the sample on a log axis. A portion of the sample tissue homogenate was used to identify and quantify individual PedsCom species by quantitative real-time PCR. Additionally, samples were compared to tissue samples from germfree control mice to further validate the assay.

[0167] 25

[0168] 4924-6583-6675, V. 1Bioinformatics and statistics. Statistical analysis was completed using Prism 10. For diabetes studies, a Log-rank test was used to compare groups. For parametric analysis of more than two groups, the ANOVA test on mean values was used with the Holm-Sidak multiple comparisons test for comparisons of all means within a test group to correct for multiple comparisons. For non-parametric analysis of more than two groups, the Kruskal-Wallis test on median values was used with post-hoc analysis consisting of the uncorrected Dunn’s test for multiple comparisons of two medians within the Kruskal- Wallis test. For the non-parametric analysis of two groups, the Mann-Whitney-Wilcoxon test on median values was used. For parametric analysis with an unequal variance of two groups, Welch’s t-test on mean values was used. For multivariate analysis, the two-way- ANOVA test with posthoc analysis consisting of the Fisher’s LSD test for direct comparisons of two means within the two-way- ANOVA test was used.

[0169] Example 2 - Results

[0170] PedsCom microbial consortium prevents T1D during an early-life window of development. Adult germfree NOD mice were colonized by oral gavage with a mixture of PedsCom microbes (109CFU of each member) (Table 1). After the initial colonization, NOD mice vertically transferred all nine of the PedsCom microbes to their progeny over multiple generations. PedsCom covers >90% of the microbial reads across the small intestine, cecum, and large intestine and mirrors the phylogenetic diversity and functions of a complete preweaning microbiome (Lubin et al., 2023). As expected, the two Lactobacillaceae species predominated in the small intestine while Parabacteroides distasonis is the most abundant microbe in the cecum and large intestine (FIGS. 1A-B). The inventors also generated the Complex Mature Community (CMCom), a complex community derived from the cecal contents of a 6-week-old Eal6 / NOD mouse to model the adult microbiome. As expected, CMCom mice microbiomes are dominated by adult-associated microbes and have a low abundance (-10%) of the early-life associated PedsCom taxa (FIGS. 6A-B). Germfree, PedsCom-, and CMCom-colonized NOD mice were bred and housed in separate gnotobiotic isolators to maintain their gnotobiotic communities.

[0171] To determine the extent to which early-life microbes derived from diabetes-protected Eocl6 / NOD mice impact the development of T1D in diabetes-susceptible mice, the inventors compared the incidence of diabetes in PedsCom-colonized NOD mice to the incidence of diabetes in germfree NOD mice and in CMCom-colonized NOD mice. Since early-life

[0172] 26

[0173] 4924-6583-6675, V. 1exposure to commensal microbes shapes immune system development, they used PedsCom and CMCom NOD mice that received their microbiota by natural vertical transmission from their dam. The incidence of diabetes in female PedsCom NOD mice was significantly lower than in germfree NOD mice (FIG. 1C; 26.3% vs 60.0%, p<0.05), demonstrating that PedsCom microbes provide protection from developing diabetes. Remarkably, PedsCom microbes also decreased the incidence and delayed the onset of diabetes compared to CMCom NOD mice (FIG. 1C; 26.3% vs 63.1%. p<0.01). Thus, colonization from birth with the nine early-life PedsCom microbes provides greater protection from diabetes than an entire complex adult-derived intestinal community. The lower diabetes incidence in PedsCom-colonized NOD mice compared to germfree and CMCom NOD mice demonstrates that this nine-member bacterial consortium is enriched for commensal microbes that are adept at preventing T1D.

[0174] The timing of microbial exposures during immune ontogeny regulates critical components of the immune system that have long-term impacts on the risk of developing inflammatory and allergic diseases (Al Nabhani et al., 2019; 2020; Knoop et al., 2020). As such, the inventors directly tested whether early-life colonization with PedsCom microbes is required for microbial protection from T1D. Germfree 6-week-old female NOD mice were cohoused with PedsCom mice to generate adult PedsCom NOD mice that had not been colonized with PedsCom microbes during early life (FIG. 7). Remarkably, these NOD mice colonized by PedsCom after weaning developed T1D at a significantly higher rate than NOD mice colonized by vertical transfer from their dams (75.0% vs 26.3%, p<0.01) (FIG. 1C). The incidence of T1D in adult-colonized mice is similar to that of NOD mice colonized with CMCom microbiota at birth (63.1%) or maintained germfree (60.0%) (FIG. 1C). Importantly, the loss of protection in adult-colonized PedsCom mice was not due to differences in microbial colonization, as adult-colonized PedsCom NOD mice possessed similar proportions of microbes compared to mice born to PedsCom-colonized dams (FIG. ID). These findings directly demonstrate that PedsCom microbes exert their protective effect during the tolerogenic window around weaning.

[0175] PedsCom colonization induces weaning-associated peripheral regulatory T cells and restrains IFNg in the pancreatic islets. To determine the immunologic impacts of PedsCom colonization in NOD mice, the inventors investigated CD4+T cell populations in gut tissues and systemic sites relevant to T1D. Specific microbes and microbial communities restrain inflammation through the induction of regulatory CD4+T cells (Atarashi et al., 2013; Arpaia et al., 2013). Microbe-induced pTregs that develop during the 3rdand 4thweeks of life are hallmarks of weaning-associated immune development in the murine gut. They are 27

[0176] 4924-6583-6675, V. 1identified by expression of transcription factors (Foxp3+, RORg+, and Helios ) and are most abundant and well-studied in the intestinal lamina propria (Sefik et al., 2015; Ohnmacht et al., 2015). More recently, pTregshave been reported to restrain insulitis (Schuster et al., 2018; Holohan et al., 2019) and the development of T1D (Schuster et al., 2018) in NOD mice.

[0177] PedsCom colonization induced a significantly higher proportion of pTregs in the lamina propria of the cecum and large intestine, the mesenteric and pancreatic lymph nodes (MLNs and PLNs), and the spleen compared to germfree NOD mice (FIGS. 2A-B and FIG. 8A), indicating that PedsCom stimulates the development of pTregsin the gut and systemic sites. When compared to CM Com-colonized mice, PedsCom microbes induced a similar proportion of pTregs in the PLNs and MLNs and a lower proportion of pTregs in the cecum and large intestine (FIGS. 2A-B and FIG. 8A). Based on the development of pTregsin the gut and systemic sites, the inventors conclude that PedsCom colonization stimulates both intestinal and systemic immune responses. Since PedsCom mice have comparable or lower levels of intestinal pTrcgsthan CMCom mice, the relative abundance of intestinal pTregs is unlikely to account for the different incidences of T1D in germfree, PedsCom, and CMCom NOD mice.

[0178] The inventor next compared IL- 10 production, as a marker of T cell suppressive function, in the gut and the PLNs of germfree, PedsCom, and CMCom NOD mice. IL- 10 restrains the development of T1D via Foxp3 -positive TregSand Foxp3-negative type 1 regulatory (Tri) cells (Yu et al., 2017; Clemente-Casares et al., 2016). Since specific commensal microbes robustly induce IL- 10 production (Jeon et al., 2012; Round and Mazmanian et al., 2010; Atarashi et al., 2011), the inventors investigated the extent to which PedsCom colonization impacts IL- 10 secretion from these regulatory cell populations in gut-associated and systemic sites relevant to T1D. Immune cells from germfree, PedsCom, and CMCom NOD mice were stimulated with phorbol myristate acetate (PMA)Zionomycin and then intracellularly stained for IL- 10. PedsCom microbes induced a higher proportion of intestinal IL-10-producing CD4+T cells and Tregscompared to GF mice and a similar proportion of intestinal IL-10-producing CD4+Tregscompared to CMCom mice (FIG. 2C and FIGS. 8B-C). In the gut, microbe-induced pTregsare the major producers of IL- 10 since most of the IL-10-producing Tregsexpressed the transcription factor RORy (FIGS. 8B-C). Overall, these findings demonstrate that PedsCom microbes more robustly induce Foxp3-positive CD4+T cells that produce the regulatory cytokine IL-10 in the PLNs that CMCom mice. (FIG. 2C). This is notable as the PLN is a critical regulatory site for the initiation and progression of islet autoimmunity (Gagnerault et al. , 2002; Gearty et al. , 2022) and suggests that PedsCom induced

[0179] 28

[0180] 4924-6583-6675, V. 1IL- 10 is well positioned to restrain autoreactive immune responses in the PLN and pancreatic islet. IL-10 counterbalances Thl-driven autoimmunity (Petrich de Marquesini et al., 2010; Arif et al., 2004), in which IFNyfrom CD4+T cells sensitizes beta cells for CD8+T-cell -mediated death (Wang et al., 1997; Yi et al., 2012).

[0181] Since PedsCom microbes robustly induced IL-10-producing Foxp3 -positive CD4+T cells, the inventors next explored the extent to which this microbial consortium induces Tri cells, Foxp3-negative, IL-10-producing CD4+T cells. Intestinal Tri cells are induced by commensal microbes, migrate to the pancreas, decrease CD4+IFNy, and delay the onset of T1D in NOD mice through the actions of the IL- 10 receptor (Yu el al., 2017; Clemente-Casares etal., 2016). PedsCom mice had a higher proportion of Tri cells in the gut and PLNs compared to CMCom mice (FIG. 2D). These results demonstrate that PedsCom microbes promote the development of both Tri cells and IL-10-producing Foxp3-positive TregSin the gut and PLNs.

[0182] Having established that PedsCom microbes induce IL-10-producing-regulatory-T cells in the gut and PLNs, the inventors hypothesized that PedsCom would decrease the proportion of pro-inflammatory IFNg-producing CD4+T cells in the pancreatic islets. Indeed, PedsCom mice had a dramatically lower proportion of IFNg-producing CD4+T cells in the pancreatic islets compared to CMCom mice (FIGS. 2E-F, 14.1% vs. 40.6%, p<0.05). This restraint of IFNy-producing CD4+T cells in PedsCom NOD mice is evident when inflammation in the islets is fully developed but before the onset of clinical signs of T1D. As such, the increased proportion of IFNg-producing CD4+T cells in CMCom islets is consistent with more pathogenic islet autoimmunity. Indeed, PedsCom and CMCom mice have similar levels of insulitis by histopathology at 10 weeks of age, despite striking differences in the risk for progression to T1 D (FIG. 9). Together, these findings provide evidence that PedsCom microbes induce regulatory cell populations, including higher proportions of IL-10-producing-Tregs and -Tri cells compared to CMCom, restrains Thl inflammation in the pancreatic islets, and prevent progression from insulitis to diabetes (FIG. 10).

[0183] Parabacteroides distasonis, Anaerostipes sp., and Clostridium intestinale induce regulatory T cells and are required to prevent T1D. One of the strengths of the PedsCom gnotobiotic model is the ability to deconstruct the community and determine the impact of each member on the development and function of the immune system. The inventors hypothesized that specific PedsCom microbes induce regulatory cells that restrain autoimmunity. To test this hypothesis, 3-week-old germfree mice were orally gavaged with 108CFUs of cultured individual PedsCom members (FIG. 3A). After two weeks, the inventors assessed the

[0184] 29

[0185] 4924-6583-6675, V. 1proportion of pTregs in colonized mice. Monocolonization with specific microbes (P. distasonis and Anaerostipes sp.) induced pTregs in the large intestine, cecum, and MLNs in NOD mice. In addition, C. intestinale and K. cowanii induced a modest but significant increase in the proportion of pTregsin the cecum (FIGS. 3B-C). The other five PedsCom microbes did not induce pTregsin monocolonized mice. These experiments provide compelling evidence that P. distasonis and Anaerostipes sp. are the strongest in vivo inducers of intestinal pTregsin PedsCom-colonized NOD mice.

[0186] To further interrogate how PedsCom colonization impacts the immune system, the inventors investigated the systemic antibody responses against each PedsCom member. Since T cells are required to develop systemic IgGl antibodies against commensal microbes (Ansaldo et al., 2019), they hypothesized that binding of the IgGl subclass to specific PedsCom species would identify those microbes capable of inducing other T-cell responses, such as induction of pTregs. Since commensal microbes also induce T-cell-independent systemic IgG subclasses (IgG2b and IgG3) (Koch et al., 2016), the inventors investigated whether PedsCom species induced these systemic antibodies as well. They perfomied microbial flow cytometry (mFLOW) to determine the degree to which IgG subclasses bind to pure cultures of each PedsCom member (Ansaldo et al., 2019; Koch et al., 2016; Moor et al., 2016)). To control for non-specific binding, they also tested sera from germfree and RAG2 deficient mice (FIG. 3D- F, FIG. 11). PedsCom colonization induced IgGl antibodies that bound to Lactobacillus johnsonii, Staphylococcus xylosus, and Mammaliicoccus sciuri (formerly Staphylococcus sciuri) (FIG. 3E). Enterococcus faecalis demonstrated inconsistent IgGl binding. Mammaliicoccus sciuri and K. cowanii elicited anti-commensal IgG2b antibodies (FIG. 3F). Anti-commensal IgG3 antibodies were not detected in PedsCom mice (FIG. 11). In contrast, the inventors did not detect any systemic antibodies against P. distasonis, Anaerostipes sp., or C. intestinale. Of note, they were unable to assess Ligilactobacillus murinus by this assay due to high nonspecific binding. In total, PedsCom-colonized NOD mice generate robust anticommensal IgGl and IgG2b antibodies against Lactobacillus johnsonii, Staphylococcus xylosus, K. cowanii, and Mammaliicoccus sciuri. In contrast to the inventors’ initial hypothesis, the PedsCom microbes that induced pTregs(P. distasonis, Anaerostipes sp. and C. intestinale) did not induce any systemic anti-commensal antibodies (FIGS. 3G-H). These data support a model in which a subset of microbes induce systemic IgG antibodies and a non-overlapping subset of PedsCom microbes induce pTregs.

[0187] Another powerful feature of the PedsCom gnotobiotic model is the ability to determine the impact of specific microbes by reconstructing the community without those microbes.

[0188] 30

[0189] 4924-6583-6675, V. 1Since P. distasonis, Anaerostipes sp., and C. intestinale induced pTregS, did not induce systemic antibodies, and are epidemiologically associated with T1D (9, 50-53), the inventors hypothesized that one or more of these three microbes is necessary for the PedsCom consortium to protect from diabetes. To directly test this hypothesis, they generated a new six-member community, PedsCom-6, that lacks P. distasonis, Anaerostipes sp., and C. intestinale (FIG. 4A and FIG. 12). The inventors then compared the incidence of T1D between PedsCom-6, PedsCom, and CMCom NOD mice. PedsCom-6 completely lost the protective qualities of the nine-member PedsCom consortium (FIG. 4B). This loss-of-function gnotobiotic experiment demonstrates that P. distasonis, Anaerostipes sp., and / or C. intestinale are required for the protection from T1D conferred by PedsCom microbes.

[0190] Early life imprinting of PD-1 pathway is required for early life microbial protection from T1D. The inventors reasoned that the PedsCom microbes induce protective immune responses early in life by upregulating tolerogenic pathways and / or downregulating the potency of autorcactivc immune cells. As such, they compared the immune profiles of PedsCom and PedsCom-6 mice at 6 weeks of age, a time period when priming of autoreactive CD4 and CD8 cells in the PLNs is important for the development of T1D. As expected, based on the monocolonization experiments (FIG. 3B-C), PedsCom mice had a modestly higher proportion of Foxp3+ Tregs in the PLNs than PedsCom-6 mice (FIG. 4C-D). In addition, CD1 Ic-i- antigen presenting cells expressed high levels of MHCII in PedsCom mice in the PLN (FIGS . 4E-F) . Unexpectedly, the inventors found a striking upregulation of inhibitor receptors PD-1 and CTL4 on CD44+ effector T CD4 and CD8 cells in the spleen, PLN, and MLN (FIG.

[0191] 4G-I). To determine the degree to which upregulation of PD-1 is required for protection from T1D in PedsCom mice, they tested whether anti-PD-1 antibody treatment would disrupt microbial protection and induce T1D in PedsCom mice. Indeed, PD-1 blockade administered to 6 week-old PedsCom mice rapidly reversed PedsCom protection and induce diabetes, with similar degree and kinetics of T1D as PedsCom-6 (FIGS. 4J-K).

[0192] Weaning-associated microbial translocation induces distinct immune responses.

[0193] Since PedsCom microbial protection from T1D requires early-life exposure, the inventors investigated whether PedsCom microbes induce diabetes-protective immune responses by gaining access to extra-intestinal sites during the tolerogenic window around weaning. They hypothesized that commensal bacteria translocate from the gut at weaning since young mice are still developing components of the intestinal barrier such as endogenous IgA (Rogier et al. , 2014), M cells (Zhang et al., 2014), and the intestinal mucus layer (Pandey et al., 2023). While a dysfunctional intestinal barrier contributes to the development of T1D in some contexts 31

[0194] 4924-6583-6675, V. 1(Sorini et al., 2019), there is limited direct evidence that a “leaky gut’’ early in life initiates this autoimmune disease. In contrast, several studies demonstrate that early-life commensal microbes induce immune responses, such as pTregs(Knoop et al., 2017; Nutsch et al., 2016) and mucosal IgA (Kramer and Cebra, 1 95), which are important for developing tolerance and preventing autoimmunity. The inventors aseptically collected and cultured tissue homogenates from the MLNs, liver, and spleen, which represent routes of lymphatic, portal vein, and systemic translocation, respectively (Berg, 1995; Balmer et al., 2014). Live PedsCom microbes were recovered from these three tissues at weaning (FIG. 5A). The MLNs were the most common site of translocation, with 44% of weanlings exhibiting bacterial translocation of up to KP CFU / gram (FIG. 5A). In contrast, culturable bacteria were not recovered at these sites from 2-week-old PedsCom mice, suggesting that weaning-associated processes permit translocation of viable commensal microbes. While bacteria could be detected in the MLN of adult mice, no bacteria were found in the spleen and liver after weaning, further suggesting a time-limited window for translocation. Furthermore, the inventors determined that there was increased translocation in the MLN of SPF C57B1 / 6 mice independent of IgA at weaning compared to post-weaning (FIG. 13). Together these findings demonstrate that there is translocation of viable bacteria from the gut to systemic sites at weaning occurs under normal physiological conditions.

[0195] To determine which PedsCom members translocate from the gut to the MLN, the inventors evaluated MLN homogenates using qPCR probes specific for the RNA polymerase beta subunit (RpoR) gene of each of the nine PedsCom bacteria. To ensure that intestinal samples were not contaminated during processing, they included MLNs from germfree mice as negative controls. At weaning, P. distasonis, Anaerostipes sp., L. johnsonii, and L. murinus DNA was recovered from the MLNs of PedsCom NOD mice (FIG. 5B). Of the PedsCom microbes that translocated to the MLNs, P. distasonis, and Anaerostipes sp. strongly induced pTreg responses but did not induce any detectable systemic antibodies, whereas L. johnsonii induced systemic IgGl antibodies but did not induce pTregs. These findings argue that bacterial translocation to systemic sites at weaning presents an opportunity for specific early-life microbes to directly influence systemic immune development. The inventors posit that the translocation of P. distasonis and Anaerostipes sp. at weaning shapes immune ontogeny toward a regulatory state and away from autoimmunity.

[0196] Finally, the inventors generated data demonstrating that PedsCom microbes can prevent another autoimmune disease. Sjogren’s disease and specifically showed that PedsCom microbes decrease the amount of inflammation in salivary glands (FIGS. 14A-B). In addition,

[0197] 32

[0198] 4924-6583-6675, V. 1they generated data demonstrating that PedsCom microbes can prevent the development of behavioral abnormalities, including decreased sociability and anxiety, in a mouse model of Autism Spectrum Disorder (ASD) (FIGS. 15A-B).

[0199] 33

[0200] 4924-6583-6675, V. 1Table 1. Pediatric Community (PedsCom)

[0201] <

[0202]

[0203]

[0204] 4924-6583-6675, V 1Table 2. Accession Numbers for Community Genome Sequences (PedsCom)

[0205] SPECIES ACCESSION NO. DATE Anaerostipes sp. PC18 GCA_039789045.1 24-M ay-24 Clostridium intestinale GCA_035905475.1 19-Jan-24 Enterococcus faecalis GCA_039791395.1 24-M ay-24 Kosakonia cowanii GCA 035928145.1 19-Jan-24 Lactobacillus johnsonii GCA 029662885.1 10-Apr-23 Ligilactobacillus murinus GCA_035904565.1 19-Jan-24 Mammaliicoccus sciuri GCA_039790465.1 25-M ay-24 Parabacteroides distasonis GCA_039789235.1 25-M ay-24 Staphylococcus xylosus GCA_039789035.1 25-M ay-24

[0206]

[0207] 4924-6583-6675, V 1Example 3 - Discussion

[0208] The inventors recently developed PedsCom, a rationally designed consortium of nine bacteria derived from the pre- weaning microbiota of diabetes -protected Eal6 / N0D mice to model the impacts of early-life microbes on immune ontogeny. In this study, the inventors investigate whether this defined consortium of microbes restrains diabetes-prone NOD mice from developing autoimmune diabetes. In total, they identified a defined group of commensal microbes that prevents T1D, delineated a developmental window for this protection, and posit that this microbially-mediated protection operates via upregulation of inhibitory receptors.

[0209] PedsCom is the first defined microbial community that protects diabetes-susceptible NOD mice from disease. Previous studies demonstrated that microbes from complex undefined commensal communities can prevent the development of T1D (Livanos et al., 2016; Markle et al., 2013: Zhang et al., 2021). However, neither well-studied gnotobiotic communities such as altered Schaedler flora (ASF) (Wen et al., 2008) nor monocolonization with immunomodulatory microbes such as segmented filamentous bacteria (SFB) elicit protection from T1D (Yurkovestskiy et al., 2013). PedsCom provides a new tool to determine how commensal microbes prevent T1D. An important feature of the gnotobiotic model is that all microbial components are defined, which allows microbes to be added or removed from the community thereby defining causal relationships between specific microbes, immune populations, and disease. As proof of principle, the inventors determined which PedsCom microbes induce regulatory T cells, and by removing these immunomodulatory microbes (P. distasonis, Anaerostipes sp., and C. intestinale), they generated a new defined community, which they called PedsCom-6. The inventors predicted that the removal of the microbes which induce pTregs would abrogate protection from T1D. Indeed, PedsCom-6 mice developed a high incidence of T1D. This microbial loss-of-function study directly demonstrates that P. distasonis, Anaerostipes sp„ and / or C. intestinale are required for microbial protection from T1D.

[0210] Many of the early events in autoimmunity begin around weaning, including the initial priming of islet autoreactive CD4+T cells, early immune cell infiltration of the islets (Gioia et al., 2019; Carrero et al., 2013; Mohan et al., 2017; Katz et al., 1993), and the development of regulatory cells such as pTregs (Sefik et al., 2015; Ohnmacht et al., 2015). Understanding what initiates and regulates this balance is critical for developing therapies to prevent T1D. The inventors directly demonstrated that PedsCom-mediated protection from T1D is dependent on early-life commensal interactions, suggesting that there are critical microbially-driven immune 36

[0211] 4924-6583-6675, V. 1interactions during this window of opportunity that restrain autoimmunity. This finding provides strong support for a causal link between specific microbes and protection from T1D, which is completely dependent on early-life exposure to these microbes. This model is consistent with human epidemiology studies of children at risk for developing T1D. For example, children who later become diabetic or develop islet autoantibodies have altered composition and functions of their gut microbiomes compared to children who do not develop T 1 D (Lubin et al. , 2019; V atanen et al. , 2018), suggesting an influence of commensal microbes on the pathogenesis of TIB (Clausen et. al., 2016; Knip and Siljander, 2016). In these studies, some microbes are associated with higher risk of autoimmunity while others are associated with lower risk of disease (Kostic et al., 2015; Stewart et al., 2018). Intriguingly, Parabacteroides distasonis has, in some studies (Stewart et al., 2018; Girdhar et al., 2022; Yue et al., 2022; Matos et al, 2021), been associated with a higher risk of developing T1D, yet was associated with protection in the inventors’ findings. One potential explanation is that timing of exposure to immunomodulatory microbes such as P. distasonis influences the trajectory of immune development toward tolerance or autoimmunity.

[0212] T1D results from an imbalance between regulatory and autoimmune processes in diabetes-susceptible individuals. Introducing PedsCom microbes into diabetes-susceptible NOD mice revealed several mechanisms by which early-life commensal microbes shifts this balance to prevent the development of T1D. PedsCom microbes shape mucosal and systemic immune development toward tolerance by inducing regulatory T cells and potently restraining inflammation in the pancreatic islets. Specifically, the inventors found that PedsCom colonization leads to an increase in two key IL-10-producing regulatory T-cell subsets (pTrcgs and Tri cells) that restrain insulitis (Schuster et al., 2018; Ilolohan et al., 2019) and T1D (Schuster et al., 2018; Yu et al., 2017; Clemente-Casares et al., 2016). PedsCom induces these cells more robustly than CMCom in the pancreatic lymph nodes (PLNs), which are a critical site for the initiation of islet autoimmunity and progression to T1D (Gagnerault et al., 2002; Gearty et al. 2022)). The pancreatic islets of PedsCom NOD mice contained far fewer IFNg-producing effector CD4+T cells than CMCom mice. Taken together, these findings argue that PedsCom microbes induce regulatory cell populations in the gut and PLNs that then restrain the accumulation of inflammatory T cells in the pancreatic islets. In support of this model, intestinal Tri cells, which can migrate to the pancreatic islets and restrain the development of T1D in an IL-10-dependent manner (Yu et al., 2017), were increased in PedsCom mice compared to CMCom mice. Furthermore, as stated earlier, the removal of the strongest pTreg-inducing species in PedsCom-6-colonized NOD mice led to the loss of protection from T1D in 37

[0213] 4924-6583-6675, V. 1Peds Com-colonized NOD mice. Unexpectedly. PedsCom mice had elevated levels of inhibitory receptors on activated CD4 and CD8 T cells which appear required to maintain microbial protection from T1D in NOD mice. The inventors propose that immunoregulatory cells traffic from the gut to the PLNs and pancreatic islets, where they oppose immune-mediated destruction of the insulin-producing beta cells, thereby preventing the development of TID.

[0214] Unexpectedly, the inventors found that P. distasonis and Anaerostipes sp. translocate to the MLN at weaning and induce pTregswithout inducing systemic antibody responses. Gut barrier dysfunction, or a “leaky gut,” has been proposed to contribute to the pathogenesis of autoimmune diabetes based on murine Joesten et al., 2019; Sorini et al., 2019) and human studies of diabetic and prediabetic subjects (Bosi et al., 2006: Li and Atkinson, 2015), yet the impacts of commensal translocation during early life on the development of T1D remain unclear. Live microbes at non-barrier sites typically provoke inflammation, yet here the inventors find that specific microbes that translocate at weaning in apparently healthy NOD mice induce the development of regulatory T cells in intestinal and, to a lesser extent, in systemic tissues. One possible explanation is that the host is permissive of low-level translocation of non-pathogenic microbes at weaning as a means to educate the immune system and develop tolerance toward endogenous commensal microbes. These findings imply that a “leaky gut” at weaning may be a physiologic mechanism to develop tolerance to commensal microbes which may have implications for other diseases such as inflammatory bowel disease. Indeed, previous studies demonstrate that microbial antigen translocation and presentation at weaning shape healthy immune development in the gut (Knoop et al., 2017), MLNs (Macpherson and Uhr, 2024), and thymus (Zegarra-Ruiz et al., 2021). During a short developmental stage around weaning, microbes preferentially induce Foxp3+RORg+pTregs, establishing peripheral tolerance to commensal microbes (Knoop et al., 2017; Nutsch et al., 2016; Lathrop et al., 2011) This “tolerogenic window” is regulated by the complex interaction of microbial and host factors, including increasing microbiota density, changes in intestinal antigen uptake, and the development of a newly described subset of intestinal APCs (Hornef et al., 2020: Knoop et al., 2017; Akagbosueta / ., 2022). In addition, a growing body of research indicates that microbial translocation influences immunity outside of the gut in the context of systemic autoimmunity (Manfredo et al., 2018) and anticancer therapies (Choi et al., 2023; Bender et al., 2023; McPherson et al., 2021). In summary, specific early-life bacteria (P. distasonis and Anaerostipes sp.) translocate from the gut at weaning, drive tolerogenic immune responses, and contribute to early-life microbial protection from T1D. These unanticipated 38

[0215] 4924-6583-6675, V. 1findings argue that the translocation of commensal microbes at weaning induces context-dependent immune responses, with consequences that likely depend upon the route and timing of translocation, intrinsic features of the translocating microbes, and / or the tissue environment in which immune cells and translocating microbes interact (McPherson et al., 2021). Based on these findings, the inventors propose that microbial re-localization during a pivotal developmental window may help establish tolerance and promote protection from autoimmunity.

[0216] In summary, the inventors leveraged a simple consortium of nine pre-weaning microbes to provide a framework for understanding how factors that shape the intestinal microbiome early in life, such as mode of birth, diet, and antibiotic treatments, may alter the risk of developing autoimmunity later in life (Jain, 2020). These results provide compelling evidence that early-life microbial interventions targeting a critical time window of immune ontogeny have the potential to prevent autoimmunity in susceptible populations.

[0217] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

[0218] 39

[0219] 4924-6583-6675, V. 1VI. References

[0220] The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

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[0322] 4924-6583-6675, V. 1

Claims

WHAT IS CLAIMED IS:

1. A method of immune modulation in a subject comprising orally administering to said subject a composition comprising Clostridium intestinale, Anaerostipes sp., and Parabacteroides distasonis.

2. A method of treating a subject with Autism Spectrum Disorder comprising orally administering to said subject a composition comprising Clostridium intestinale, Anaerostipes sp., and Parabacteroides distasonis.

3. The method of claim 1 or claim 2, wherein the modulation results in reducing the risk of developing autoimmunity or the degree or extent of autoimmunity in said subject.

4. The method of any one of claims 1-3, further comprising administering to said subject Lactobacillus johnsonii, Ligilactobacillus murinus, Mammaliicoccus sciuri, Kosakonia cowanii, Enterococcus faecalis, and Staphylococcus xylosus.

5. The method of any one of claims 1-4, wherein said subject is less six months old or less, 3 months or less, 2 months old or less, 6 weeks old or less, 1 month old or less, or 4 weeks old or less.

6. The method of any one of claims 1-5, wherein the subject is a human subject, a murine subject, or a mammalian subject.

7. The method of any one of claims 1-6, wherein the composition is administered more than once, such as two, three, four, five, six, seven, eight, nine, ten, twelve, fifteen or twenty times.

8. The method of claim 7, wherein the composition is administered daily, every other day, every third day, every four days, even week, every other week, or monthly.

9. The method of any one of claims 1 or 3-7, wherein the reduced or diminished risk of autoimmunity relates to celiac disease, diabetes mellitus type 1, Henoch-Schonlein purpura, systemic lupus erythematosus, Sjogren syndrome, eosinophilic granulomatosis with polyangiitis, Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, Addison's disease, rheumatoid arthritis, ankylosing spondylitis, polymyositis,464924-6583-6675, V. 1dermatomyositis, multiple sclerosis, inflammatory bowel disease (e.g. Crohn’s disease, ulcerative colitis), or checkpoint inhibitor induced autoimmunity.

10. The method of any one of claims 1 and 3-9, wherein said subject has a genetic, environmental or therapy-related predisposition to developing autoimmunity.

11. The method of any one of claims 1 and 3-10, wherein the method results in one or more of (a) induced PD-1 expression, (b) induced IL- 10 production, (c) induced CTLA-4 expression, and / or (c) induced peripheral regulatory T cells, such as CD4 and CD8 T cells.

12. The method of claim 11, further comprising assessing one or more of (a) induced PD-1 expression, (b) induced IL- 10 production, (c) induced CTLA-4 expression, and / or (c) induced peripheral regulatory T cells, such as CD4 and CD8 T cells prior to and / or after administering.

13. The method of claim 1, wherein the method results in a reduced severity of type 1 diabetes in the subject.

14. The method of any one of claims 1-13, wherein the subject is a human subject.

15. The method of any one of claims 1 and 3-14, wherein the subject or human subject is treated with insulin.

16. The method of any one of claims 1 and 3-15, wherein the subject or human subject exhibits one or more of improved glucose regulation, reduced A1C scores, and / or improved lipid profile.

17. The method of any one of claims 2-12 and 14, wherein the administration results in improvement of one or more of challenges with social communication / interaction (e.g., poor eye contact, difficulty with back-and-forth conversation), restricted, repetitive patterns of behavior or interests (e.g., hand-flapping, intense focus on specific topics, strict routines), sensory sensitivities (e.g., to light, sound, touch), anxiety, unusual eating patterns, and / or unusual sleeping patterns474924-6583-6675, V. 1

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