Relevance of lymphocyte proliferation to PHA in severe combined immunodeficiency (SCID) and T cell lymphopenia

Roshini S Abraham¹, Amrita Basu², Jennifer R Heimall³, Elizabeth Dunn⁴, Alison Yip⁴, Malika Kapadia⁵, Neena Kapoor⁶, Lisa Forbes Satter⁷, Rebecca Buckley⁸, Richard O'Reilly⁹, Geoffrey D E Cuvelier¹⁰, Sharat Chandra¹¹, Jeffrey Bednarski¹², Sonali Chaudhury¹³, Theodore B Moore¹⁴, Hilary Haines¹⁵, Blachy J Dávila Saldaña¹⁶, Deepakbabu Chellapandian¹⁷, Ahmad Rayes¹⁸, Karin Chen¹⁹, Emi Caywood²⁰, Shanmuganathan Chandrakasan²¹, Mark Thomas Vander Lugt²², Christen Ebens²³, Pierre Teira²⁴, Evan Shereck²⁵, Holly Miller²⁶, Victor Aquino²⁷, Hesham Eissa²⁸, Lolie C Yu²⁹, Alfred Gillio³⁰, Lisa Madden³¹, Alan Knutsen³², Ami J Shah³³, Kenneth DeSantes³⁴, Jessie Barnum³⁵, Larisa Broglie³⁶, Avni Y Joshi³⁷, Gary Kleiner³⁸, Jasmeen Dara⁴, Susan Prockop⁵, Caridad Martinez⁷, Talal Mousallem⁸, Joseph Oved⁹, Lauri Burroughs¹⁹, Rebecca Marsh³⁹, Troy R Torgerson¹⁹, Jennifer W Leiding⁴⁰, Sung Yun Pai⁴¹, Donald B Kohn⁴², Michael A Pulsipher¹⁸, Linda M Griffith⁴³, Luigi D Notarangelo⁴⁴, Morton J Cowan⁴, Jennifer Puck⁴, Christopher C Dvorak⁴, Elie Haddad²⁴

Affiliations

¹ Department of Pathology and Laboratory Medicine, Nationwide Children's Hospital, OH, USA. Electronic address: Roshini.Abraham@nationwidechildrens.org.
² Department of Pathology and Laboratory Medicine, Nationwide Children's Hospital, OH, USA.
³ Division of Allergy and Immunology, Children's Hospital of Philadelphia, PA, USA.
⁴ Division of Allergy, Immunology, and Blood and Marrow Transplantation, Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA.
⁵ Division of Pediatric Oncology, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Department of Pediatrics, Harvard University Medical School, Boston, MA, USA.
⁶ Transplantation and Cellular Therapy Program, Cancer and Blood Disease Institute, Children's Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
⁷ Pediatrics, Immunology, Allergy and Rheumatology, Baylor College of Medicine, Houston, TX, USA.
⁸ Departments of Pediatrics and Immunology, Duke University Medical Center, Durham, NC, USA.
⁹ Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
¹⁰ Manitoba Blood and Marrow Transplant Program, CancerCare Manitoba, Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, Canada.
¹¹ Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA; Division of Bone Marrow Transplantation and Immune Deficiency, Cancer and Blood Disease Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.
¹² Division of Hematology and Oncology, Department of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA.
¹³ Division of Hematology, Oncology, and Stem Cell Transplantation, Ann & Robert H. Lurie Children's Hospital of Chicago-Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
¹⁴ Division of Hematology/Oncology, Mattel Children's Hospital at UCLA, Los Angeles, CA, USA.
¹⁵ Division of Pediatric Hematology-Oncology and Bone Marrow Transplant, University of Alabama at Birmingham, Birmingham, AL, USA.
¹⁶ Division of Blood and Marrow Transplantation, Children's National Hospital-George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
¹⁷ Blood and Marrow Transplant, Johns Hopkins All Children's Hospital, St. Petersburg, FL, USA.
¹⁸ Division of Pediatric Hematology and Oncology, Intermountain Primary Childrens Hospital, Huntsman Cancer Institute, Spencer Fox Eccles School of Medicine at the University of Utah, Salt Lake City, UT, USA.
¹⁹ Department of Pediatrics, University of Washington-Seattle Children's Hospital, Seattle, WA, USA.
²⁰ Nemours Children's Health Delaware, Thomas Jefferson University, Wilmington, DE, USA.
²¹ Bone Marrow Transplantation Program, Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA.
²² Blood and Marrow Transplant Program, University of Michigan, Ann Arbor, MI, USA.
²³ Division of Pediatric Blood and Marrow Transplantation & Cellular Therapy, University of Minnesota, Minneapolis, MN, USA.
²⁴ Pediatric Immunology and Rheumatology Division, CHU Sainte-Justine, Department of Pediatrics, University of Montreal, Montreal, QC, Canada.
²⁵ Division of Pediatric Hematology/Oncology, Oregon Health and Science University, Portland, OR, USA.
²⁶ Phoenix Children's Hospital, Phoenix, AZ, USA.
²⁷ Division of Pediatric Hematology, Oncology and Stem Cell Transplantation, University of Texas Southwestern Medical Center, Dallas, TX, USA.
²⁸ Division of Pediatric Hematology-Oncology-BMT, University of Colorado, Aurora, CO, USA.
²⁹ Division of Pediatric Hematology-Oncology/HSCT, LSUHSC and Children's Hospital, New Orleans, LA, USA.
³⁰ Institute for Pediatric Cancer and Blood Disorders, Hackensack University Medical Center, Hackensack, NJ, USA.
³¹ Pediatric Blood and Marrow Transplantation Program, Texas Transplant Institute, Methodist Children's Hospital, San Antonio, TX, USA.
³² Department of Pediatrics, Pediatric Allergy and Immunology Division, Saint Louis University, St Louis, MO, USA.
³³ Division of Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford School of Medicine Pediatric Stem Cell Transplantation, Stanford University, Stanford, CA, USA.
³⁴ American Family Children's Hospital, University of Wisconsin, Madison, WI, USA.
³⁵ UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA, USA.
³⁶ Division of Pediatric Hematology, Oncology, and Blood and Marrow Transplantation, Medical College of Wisconsin, Milwaukee, WI, USA.
³⁷ Division of Pediatric and Adult Allergy and Immunology, Mayo Clinic, Rochester, MN, USA.
³⁸ Division of Pediatric Allergy and Immunology, Department of Pediatrics, Holtz Children's Hospital at Jackson Memorial Hospital, University of Miami Miller School of Medicine, Miami, FL, USA.
³⁹ Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA; Division of Bone Marrow Transplantation and Immune Deficiency, Cancer and Blood Disease Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Pharming Healthcare Inc, Warren, NJ, USA.
⁴⁰ Division of Allergy and Immunology, Department of Pediatrics, Johns Hopkins University Baltimore, MD and Institute for Clinical and Translational Research, Johns Hopkins All Childrens Hospital, St. Petersburg, FL, USA.
⁴¹ Immune Deficiency Cellular Therapy Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
⁴² Department of Pediatrics, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA, USA.
⁴³ Division of Allergy, Immunology, and Transplantation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
⁴⁴ Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

PMID: 38367737
PMCID: PMC11018339
DOI: 10.1016/j.clim.2024.109942

Relevance of lymphocyte proliferation to PHA in severe combined immunodeficiency (SCID) and T cell lymphopenia

Roshini S Abraham et al. Clin Immunol. 2024 Apr.

. 2024 Apr:261:109942.

doi: 10.1016/j.clim.2024.109942. Epub 2024 Feb 15.

Authors

Affiliations

¹ Department of Pathology and Laboratory Medicine, Nationwide Children's Hospital, OH, USA. Electronic address: Roshini.Abraham@nationwidechildrens.org.
² Department of Pathology and Laboratory Medicine, Nationwide Children's Hospital, OH, USA.
³ Division of Allergy and Immunology, Children's Hospital of Philadelphia, PA, USA.
⁴ Division of Allergy, Immunology, and Blood and Marrow Transplantation, Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA.
⁵ Division of Pediatric Oncology, Dana-Farber/Boston Children's Cancer and Blood Disorders Center, Department of Pediatrics, Harvard University Medical School, Boston, MA, USA.
⁶ Transplantation and Cellular Therapy Program, Cancer and Blood Disease Institute, Children's Hospital Los Angeles, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
⁷ Pediatrics, Immunology, Allergy and Rheumatology, Baylor College of Medicine, Houston, TX, USA.
⁸ Departments of Pediatrics and Immunology, Duke University Medical Center, Durham, NC, USA.
⁹ Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
¹⁰ Manitoba Blood and Marrow Transplant Program, CancerCare Manitoba, Department of Pediatrics and Child Health, University of Manitoba, Winnipeg, MB, Canada.
¹¹ Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA; Division of Bone Marrow Transplantation and Immune Deficiency, Cancer and Blood Disease Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA.
¹² Division of Hematology and Oncology, Department of Pediatrics, Washington University School of Medicine, St. Louis, MO, USA.
¹³ Division of Hematology, Oncology, and Stem Cell Transplantation, Ann & Robert H. Lurie Children's Hospital of Chicago-Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
¹⁴ Division of Hematology/Oncology, Mattel Children's Hospital at UCLA, Los Angeles, CA, USA.
¹⁵ Division of Pediatric Hematology-Oncology and Bone Marrow Transplant, University of Alabama at Birmingham, Birmingham, AL, USA.
¹⁶ Division of Blood and Marrow Transplantation, Children's National Hospital-George Washington University School of Medicine and Health Sciences, Washington, DC, USA.
¹⁷ Blood and Marrow Transplant, Johns Hopkins All Children's Hospital, St. Petersburg, FL, USA.
¹⁸ Division of Pediatric Hematology and Oncology, Intermountain Primary Childrens Hospital, Huntsman Cancer Institute, Spencer Fox Eccles School of Medicine at the University of Utah, Salt Lake City, UT, USA.
¹⁹ Department of Pediatrics, University of Washington-Seattle Children's Hospital, Seattle, WA, USA.
²⁰ Nemours Children's Health Delaware, Thomas Jefferson University, Wilmington, DE, USA.
²¹ Bone Marrow Transplantation Program, Aflac Cancer and Blood Disorders Center, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA.
²² Blood and Marrow Transplant Program, University of Michigan, Ann Arbor, MI, USA.
²³ Division of Pediatric Blood and Marrow Transplantation & Cellular Therapy, University of Minnesota, Minneapolis, MN, USA.
²⁴ Pediatric Immunology and Rheumatology Division, CHU Sainte-Justine, Department of Pediatrics, University of Montreal, Montreal, QC, Canada.
²⁵ Division of Pediatric Hematology/Oncology, Oregon Health and Science University, Portland, OR, USA.
²⁶ Phoenix Children's Hospital, Phoenix, AZ, USA.
²⁷ Division of Pediatric Hematology, Oncology and Stem Cell Transplantation, University of Texas Southwestern Medical Center, Dallas, TX, USA.
²⁸ Division of Pediatric Hematology-Oncology-BMT, University of Colorado, Aurora, CO, USA.
²⁹ Division of Pediatric Hematology-Oncology/HSCT, LSUHSC and Children's Hospital, New Orleans, LA, USA.
³⁰ Institute for Pediatric Cancer and Blood Disorders, Hackensack University Medical Center, Hackensack, NJ, USA.
³¹ Pediatric Blood and Marrow Transplantation Program, Texas Transplant Institute, Methodist Children's Hospital, San Antonio, TX, USA.
³² Department of Pediatrics, Pediatric Allergy and Immunology Division, Saint Louis University, St Louis, MO, USA.
³³ Division of Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford School of Medicine Pediatric Stem Cell Transplantation, Stanford University, Stanford, CA, USA.
³⁴ American Family Children's Hospital, University of Wisconsin, Madison, WI, USA.
³⁵ UPMC Children's Hospital of Pittsburgh, Pittsburgh, PA, USA.
³⁶ Division of Pediatric Hematology, Oncology, and Blood and Marrow Transplantation, Medical College of Wisconsin, Milwaukee, WI, USA.
³⁷ Division of Pediatric and Adult Allergy and Immunology, Mayo Clinic, Rochester, MN, USA.
³⁸ Division of Pediatric Allergy and Immunology, Department of Pediatrics, Holtz Children's Hospital at Jackson Memorial Hospital, University of Miami Miller School of Medicine, Miami, FL, USA.
³⁹ Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA; Division of Bone Marrow Transplantation and Immune Deficiency, Cancer and Blood Disease Institute, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, USA; Pharming Healthcare Inc, Warren, NJ, USA.
⁴⁰ Division of Allergy and Immunology, Department of Pediatrics, Johns Hopkins University Baltimore, MD and Institute for Clinical and Translational Research, Johns Hopkins All Childrens Hospital, St. Petersburg, FL, USA.
⁴¹ Immune Deficiency Cellular Therapy Program, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.
⁴² Department of Pediatrics, David Geffen School of Medicine at University of California, Los Angeles, Los Angeles, CA, USA.
⁴³ Division of Allergy, Immunology, and Transplantation, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.
⁴⁴ Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA.

PMID: 38367737
PMCID: PMC11018339
DOI: 10.1016/j.clim.2024.109942

Abstract

Severe combined immunodeficiency (SCID) is characterized by a severe deficiency in T cell numbers. We analyzed data collected (n = 307) for PHA-based T cell proliferation from the PIDTC SCID protocol 6901, using either a radioactive or flow cytometry method. In comparing the two groups, a smaller number of the patients tested by flow cytometry had <10% of the lower limit of normal proliferation as compared to the radioactive method (p = 0.02). Further, in patients with CD3+ T cell counts between 51 and 300 cells/μL, there was a higher proliferative response with the PHA flow assay compared to the ³H-T assay (p < 0.0001), suggesting that the method of analysis influences the resolution and interpretation of PHA results. Importantly, we observed many SCID patients with profound T cell lymphopenia having normal T cell proliferation when assessed by flow cytometry. We recommend this test be considered only as supportive in the diagnosis of typical SCID.

Keywords: Mitogen; PHA; PIDTC; SCID; Severe combined immunodeficiency; T-cell lymphopenia; T-cell proliferation.

PubMed Disclaimer

Conflict of interest statement

Declaration of competing interest RSA has been on the advisory board for Horizon Pharma (now Amgen) and Sobi in the past 2 years but is not currently serving in that capacity. RM is an employee of Pharming Healthcare Inc., Warren, NJ. JWL is an employee and shareholder of Bluebird Bio, and has been a speaker/consultant and on the advisory board for Sobi, Inc.

Figures

**Fig. 1.**
Flow chart depicting selection of protocol # 6901 SCID patients for the study. A total of 395 individuals were screened for eligibility for this study and 35 patients were excluded by the PIDTC 6901 protocol. Of the remaining 362 patients, 55 were excluded and 307 were eligible for this study.

**Fig. 2.**
PHA induced proliferative response measured by ³H-T radioactivity-based and CD3+ T cell flow-based assays. (A) Proportion (%) of patients having ≤10% (n = 155 for ³H-T; flow: 58) and > 10% LLN (n = ³H-T: 52; Flow: 42) proliferative response to PHA measured by either method. (B) The full range of %LLN proliferation response in all patients assessed by both methods (n = ³H-T: 207; flow: 100). (C) This graph demonstrates proliferation response ≤50% LLN in patients assessed by each method. (** = p < 0.01; ns, not significant. (n = ³H-T: 188; Flow: 83). Horizontal lines represent the median of each dataset. For flow cytometry, T cells were identified using CD3 and side scatter (SSC) from the lymphocyte (CD45+/SSC) population.

**Fig. 3.**
Comparison of % LLN proliferative response by radioactive and CD3+ T cell flow-based methods relative to the T cell count. (A) CD3+ T cell count ≤50 cells/μL (n = ³H-T: 112, Flow: 62); (B) The number of patients with ≤10%LLN (n = ³H-T: 103, Flow: 49) or > 10%LLN (n = ³H-T: 9, Flow: 13) in the ³H-T vs. flow method and CD3+ T cell count ≤50 cells/μL (p value based on Fisher’s exact test); (C) the number of patients with a baseline CD3+ T cell count ≤300 cells/μL and ≤ 30% LLN (n = ³H-T: 160, Flow: 67) or > 30% LLN (n = ³H-T: 11, Flow: 24); (D) CD3+ T cell count = 51–300 cells/μL (n = ³H-T: 59, Flow: 29), (E) CD3+ T cell count >300 cells/μL (n = ³H-T:36, Flow: 9). The median of each dataset is depicted by a horizontal line.

**Fig. 4.**
PHA proliferative response in patients with or without transplacental maternal engraftment (TME). The effect of TME on the T cell proliferative response was assessed for the (A) ³H-T and (B) flow ³H-T by rank comparison of the nonparametric datasets with Mann-Whitney test. (C) Comparison between ³H-T and flow assay in SCID patients without TME, using the Mann-Whitney non-parametric test. The median of each dataset is depicted in the graph. The number of patients with TME present is 44 and with TME absent (n = 94) for ³H-T; for flow cytometry, TME present (n = 22) and TME absent (n = 62).

**Fig. 5.**
PHA proliferative response in patients with and without Omenn syndrome. The association of PHA proliferative response in Omenn syndrome (OS) and non-Omenn (nOS) patients measured by (A) radioactive and (B) flow cytometry was assessed using Mann-Whitney test for non-parametric rank comparison. The median of each dataset is depicted in the graph. (C) Distribution of %LLN proliferative response measured by radioactivity and flow-based assays relative to the CD3+ T cell count in Omenn Syndrome patients, and (D) non-Omenn patients. The rectangle represents patients with CD3+ T cell counts ≤300 cells/μL. There are 15 patients in the OS group and 192 patients in the nOS group evaluated by ³H-T. In the group evaluated by flow cytometry, there are 5 patients in the OS group and 95 patients in the nOS group.

**Fig. 6.**
Comparison of PHA proliferation for CD45+ lymphocytes or CD3+ T cells using the flow cytometry method. Data for PHA proliferation using either CD45+ lymphocytes or CD3+ T cells categorized by baseline CD3+ T cell count. (A) %LLN proliferation using either CD45+ lymphocyte (n = 58) or CD3+ T cell (n = 62) data for CD3+ T cell count ≤50 cells/μL; (B) Fisher exact test comparison of the frequency (%) of patients with either <10% LLN or > 10% LLN for CD45+ lymphocyte or CD3+ T cell data (p = 0.02); (C) %LLN proliferation using either CD45+ lymphocyte (n = 27) or CD3+ T cell (n = 29) data for CD3+ T cell count 51–300 cells/μL; (D) %LLN proliferation using either CD45+ lymphocyte (n = 8) or CD3+ T cell (n = 9) data for CD3+ T cell count >300 cells/μL.

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Relevance of lymphocyte proliferation to PHA in severe combined immunodeficiency (SCID) and T cell lymphopenia

Affiliations

Relevance of lymphocyte proliferation to PHA in severe combined immunodeficiency (SCID) and T cell lymphopenia

Authors

Affiliations

Abstract

Conflict of interest statement

Figures

References

Publication types

MeSH terms

Grants and funding

LinkOut - more resources

Full Text Sources

Medical