https://orcid.org/0009-0007-6577-687X
Figures
Abstract
Voluntary medical male circumcision (VMMC) reduces HIV acquisition by at least 60%, but the determinants of HIV susceptibility in foreskin tissues are incompletely understood. Flow cytometry is a powerful tool that helps us understand tissue immune defenses in mucosal tissue like the inner foreskin, but foreskin flow cytometry has only been validated using fresh tissue samples. This restricts immune analyses to timepoints immediately after surgical acquisition and hinders research in this area. We compared fresh analysis with whole tissue cryopreservation and later thawing and digestion to analyze CD4+ T cell populations relevant to HIV susceptibility (CCR5, CD25, CD127, CCR4, CXCR3, CCR6, CCR10, HLA-DR, and CD38). Eight foreskin samples from HIV-negative males aged >18 years were collected after VMMC. For each sample, half the foreskin was immediately cryopreserved for later digestion and flow cytometry analysis, while the remaining tissues were analyzed fresh. We demonstrate no significant impact of cryopreservation on CD4+ T cell expression of CD25, CCR4, CCR6, HLA-DR, CCR10, or CD127. Although expression levels of CCR5, CD38, and CXCR3 were increased after cryopreservation, the relative ranking of participants was retained. In conclusion, cryopreserved foreskin tissues may be suitable for subsequent digestion and flow cytometry phenotyping of HIV-susceptible T cell populations.
Citation: Almomani O, Nnamutete J, Shao Z, Biribawa VM, Ssemunywa H, Namuniina A, et al. (2024) Effect of cryopreservation on CD4+ T cell subsets in foreskin tissue. PLoS ONE 19(3): e0297884. https://doi.org/10.1371/journal.pone.0297884
Editor: Joseph J. Mattapallil, Uniformed Services University, UNITED STATES
Received: August 9, 2023; Accepted: January 3, 2024; Published: March 1, 2024
Copyright: © 2024 Almomani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting information files.
Funding: - Canadian Foundation for Innovation, John R. Evans Leaders Fund (42343) [JLP] [https://www.innovation.ca/apply-manage-awards/funding-opportunities/john-r-evans-leaders-fund] - Canada Research Chair Program (CRC-2020-00175) [JLP] [https://www.chairs-chaires.gc.ca/home-accueil-eng.aspx] - National Institute of Allergy and Infectious Diseases (R01AI128779) [AART] [https://www.niaid.nih.gov/] - National Institute of Diabetes and Digestive and Kidney Diseases (R01DK131936) [CML] [https://www.niddk.nih.gov/] - Canadian Institutes of Health Research (PJT-180629) [RK] [https://cihr-irsc.gc.ca/e/50513.html] The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Voluntary medical male circumcision (VMMC) reduces HIV acquisition in heterosexual men by at least 60% [1–3], but the mechanism(s) through which foreskin removal mediates HIV protection is not fully understood [4]. The tremendous public health benefits that VMMC programs have provided in priority countries [5,6] highlight the need for further research. Furthermore, VMMC reduces the acquisition of other viral sexually transmitted infections (STIs), including HPV and HSV-2 [1,7,8], and not only profoundly alters the penile microbiome of the host but also reduces bacterial vaginosis in female sexual partner(s) [9].
Although additional research on the immune parameters of HIV acquisition in the foreskin is needed, efforts to study foreskin immune cell populations and other immune parameters have been limited by the relative difficulty in obtaining these tissues from adults. Obtaining entire foreskin tissues during VMMC is relatively straightforward, but it is generally not feasible to obtain foreskin biopsies, which are more easily obtained at other mucosal sites such as the ectocervix, vagina, or anorectum. Therefore many published studies of HIV target cells in foreskin tissue have performed analysis immediately after circumcision using fresh samples [10–14]. Cryopreservation of foreskin tissues collected from adult males undergoing VMMC could substantially expand future research, allowing researchers to nest more sophisticated immune analyses in specific participants.
Unfortunately, there are currently no data regarding the effect of cryopreservation on foreskin T cell recovery relevant to HIV susceptibility on foreskin derived CD4+ T cells. Previous work in ectocervical and colorectal tissues demonstrated that the cryopreservation of whole tissues gives a higher cell yield and more representative T cell function than the cryopreservation of post-digestion single-cell suspensions [15]. In response to cryopreservation, the relative shifts in cell populations may be accounted for by the upregulation of particular markers [16] or the preferential death of subsets more susceptible to mechanical stress accumulated by cryopreservation [17]. Given the importance of the foreskin in HIV acquisition [4,7,18–28], we sought to investigate how cryopreservation alters the characteristics of potential CD4+ T cell targets.
Methods
Participants and sample processing
Participants were recruited from the VMMC program at Kisubi Hospital in Entebbe, Uganda. All participants were recruited in the months of August and September (2022), and participants provided informed consent verbally and in writing. Foreskin samples were collected from n = 8 HIV-negative adult males (18 years and older) with no clinically apparent genital signs or symptoms (discharge, ulcers, warts). Tissues were collected immediately after surgery and placed in a dry labeled 50 mL centrifuge tube without any transport media. Samples were without media for 15–30 minutes as they were transported from the site of VMMC to the research facility. Transport media was not used for two reasons. First, power supply is often inconsistent at sites of VMMC (including the areas surrounding Kisubi Hospital), and thus it is difficult to ensure consistent media refrigeration prior to VMMC. We therefore did not include media to ensure the methods reported in this study would be implementable in low-resources settings with inconsistent power supplies or lack of access to refrigeration. Moreover, media was avoided to maintain compatibility with studies where tissues will be later frozen for immunofluorescence (IF) microscopy. Our experience is that soaking tissues in media results in edema and tissue damage after freezing in Optimal Cutting Temperature (OCT) compound. Upon reaching the lab, the tissue was placed in a petri-dish and introduced to R10 media (RPMI with 10% HI-FBS, 10 mM HEPES buffer, 2 mM L-glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin, 100 μg/mL streptomycin (all from Sigma)) to prevent drying of the tissue. The inner and outer aspects of the foreskin were identified, and the tissue was separated into two pieces containing equal quantities of inner and outer aspects (Fig 1). A 6 cm2 piece was dissected from each half using a scalpel, preferentially containing the inner aspect and augmenting with tissue from the outer aspect only when necessary (i.e., for smaller foreskins). Each 6 cm2 piece was further sectioned into 24 pieces, each measuring 0.25 cm2 squares that were either immediately processed for flow cytometry or cryopreserved for 2–5 days before processing.
Samples were divided equally, with one portion processed for flow cytometry immediately and one cryopreserved for 2–5 days before processing. Both samples were digested to a single cell suspension by mechanical and enzymatic digestion (collagenase) in the presence of DNAse. Cell suspensions were filtered to remove debris, stained with fluorescently labeled antibodies against markers of interest, and analyzed using an LSRII flow cytometer (HI-FBS: heat-inactivated FBS). Created with BioRender.com.
Cryopreservation and thawing of tissue
Each of the 24 tissue sections for cryopreservation was placed in a cryovial containing 1mL chilled cryopreservation media (10% DMSO, 90% HI-FBS, both Sigma). Cryovials were cooled to -80°C at 1°C /min using a freezing container (Mr. Frosty, Nalgene), then transferred to the vapor phase of liquid nitrogen for 2–5 days. Following cryopreservation, cryovials were removed from liquid nitrogen and agitated gently in a 37°C water bath until just thawed. Thawed tissues were transferred to 25mL of R10 for 10 minutes to allow DMSO to elute from the tissue.
Cell isolation from foreskin tissues
Each tissue was placed in a 1.5mL snap-top tube (Eppendorf) containing 0.5mL of RPMI, and sterile surgical scissors were used to mechanically disrupt tissues as much as possible. RPMI with collagenase (Type I powder, ThermoFisher Scientific) and DNAse I (ThermoFisher Scientific) were added to each tube to a final concentration of 500U/mL and 125 units/μl, respectively. Tubes were placed on a shaker with a heating block (Eppendorf ThermoMixer) at 900rpm and 37°C for 40 minutes. After 40 minutes, the cell suspensions were pooled into a 50mL conical tube containing 3mL FBS. The resulting suspension was filtered through a 100μm filter (Cell Strainer, Fisher Scientific) and centrifuged for 6 minutes at 241g. The cell pellet was gently resuspended in 20mL R10, and the number of live (trypan blue exclusion, Sigma) mononuclear cells was estimated by light microscopy. Isolated cells were rested overnight in an incubator at 37°C and 5% CO2.
Flow cytometry
Rested cells were washed and resuspended in 1mL PBS. Cells were stained first with LIVE/DEAD Fixable Near-IR Dead Cell Stain (Invitrogen), and then with the following fluorescently labeled anti-human antibodies (S1 Table) at 4°C for 20 minutes: CD3, CD4, CD38, HLA-DR, CD25, CD127, CD194 (CCR4), CD183 (CXCR3), CD196 (CCR6), CD195 (CCR5), and CCR10. Antibodies were chosen based on their ability to differentiate T cell populations relevant to HIV susceptibility [29–32]: Th1 (CXCR3+, CCR6-), Th2 (CCR4+, CCR6-), Th17 (CCR4+, CCR6+), Th22 (CCR10+, CCR4+, CCR6+), T regulatory cells (Treg, CD25+, CD127LOW); to identify activated T cells (HLA-DR and CD38); and expression of the HIV co-receptor essential for sexual transmission (CCR5) [33,34]. Cells were washed in PBS, fixed in 200mL of 2% formalin (Zayo-Sigma) for 20 minutes at 4°C, washed, and resuspended in 200mL of PBS. Flow cytometry data were acquired on an LSR II flow cytometer (BD Biosciences) using FACSDiva Software (BD Biosciences, version 6.0). Samples were run to completion, and the total number of live/dead-negative (live) and CD3+ events obtained for each sample was recorded as a measure of T cell recovery. Fluorescence Minus One (FMO) controls were run using cells from fresh foreskin tissues and PBMCs during panel development and antibody titration. FMOs were also run at the study midpoint (on PBMCs). Identical gates were applied to fresh and cryopreserved samples from the same participant. FMOs were used to guide gating when negative and positive populations were not readily discernable. The gating strategy is shown in Fig 2.
Doublet events were excluded based on height vs. area forward scatter, and dead cells were excluded. Live singlet events were then gated based on the positive expression of CD3, and the number of events in this gate was used to calculate the total T cells recovered. Total T cells were sub-gated on CD4+ events, and expression of all other phenotype markers was determined based on this population (shown for both tissues processed immediately after VMMC (fresh) and those processed after cryopreservation). Gates for each participant’s fresh and cryopreserved samples were identical, and FMO controls were used to assist with gating when populations were not clearly distinct.
Statistical analysis
Flow cytometry gating was performed using FlowJo software (version 10, FlowJo LLC). For each participant, gates were identical between the freshly digested portion of the foreskin and the cryopreserved counterpart. The proportion of live/CD3+ events positive for CD4 and the proportion of live/CD3+/CD4+ events positive for each remaining marker were compared between fresh and cryopreserved samples. Distributions of cell counts (for total T cells recovered) or proportional abundances (of markers as a % of live/CD3+/CD4+ cells) were compared using the Wilcoxon matched-pairs signed rank test. Statistical analyses were performed using Prism (v9, GraphPad).
Ethics statement
All participants provided written informed consented to participate in this study. The study was reviewed and approved by research ethics boards at the Ugandan Virus Research Institute, the Uganda National Council for Science and Technology, the University of Toronto, and Western University.
Results
The total number of T cells recovered from fresh digestion of the foreskin was significantly higher than the number recovered from foreskin tissue that had been cryopreserved (median 9.3x103 live CD3+ events vs. 2.1x103, p <0.01, Fig 3A). Within the T cell population (CD3+), the relative abundance of T cells expressing CD4 did not differ between fresh and cryopreserved tissue (median 62% vs. 58%, p = 0.38, Fig 3B). One sample was insufficiently stained for CCR4 and thus did not contribute to the analysis of this marker (n = 7 for comparing %CCR4+ in fresh vs. cryopreserved). There were also no significant differences in the relative abundance of CD4 T cells positive for CCR4 (median 54% vs. 59%, p = 0.08), CCR6 (median 40% vs. 36%, p = 0.20, CCR10 (median 5.0% vs. 5.5%, p = 0.38), CD25 (median 12% vs. 12%, p = 0.31), CD127 (median 74% vs. 75%, p = 0.95), and HLA-DR (median 24% vs. 17%, p = 0.38, Fig 4A–4F). However, after cryopreservation, there were statistically significant increases in the relative abundance of CD4 T cells expressing CCR5 (median 54% vs. 66%, p = 0.02), CD38 (median 3.6% vs. 6.0%, p = 0.02), and CXCR3 (median 22% vs. 33%, p<0.01, Fig 4G–4I). For CCR5 and CD38, 7/8 cryopreserved samples (n = 8) exhibited increased proportions of CCR5 and CD38 expression, while all samples showed increased proportions of CXCR3 expression. However, the relative ranking of participants’ expression of these markers was relatively stable (e.g., participants who ranked among the highest expressers in fresh conditions also ranked among the highest levels after cryopreservation). Similar findings were observed when examining the proportion of total CD3+ cells (i.e., not limiting to CD3+/CD4+ cells, S1 Fig).
(A) Total T cells recovered was defined as the total number of Live/Dead-, CD3+ events detected by flow cytometry when acquiring the complete sample generated from the digestion of 6cm2 of tissue. The total number of T cells recovered was higher in tissues processed immediately (fresh) than those processed after cryopreservation. (B) The relative abundance of T cells expressing CD4 did not differ between tissues processed immediately (fresh) compared to those processed after cryopreservation. Wilcoxon matched-pairs signed rank tests were used for both analyses (n = 8), *denotes p-value <0.05, ns (non-significant) denotes p-value >0.06, exact p-value listed when 0.06>p>0.05.
There were no significant differences in the relative abundance of CD4 T cells expressing CCR4 (A), CCR6 (B), CCR10 (C), CD25 (D), CD127 (E), and HLA-DR (F) between tissues processed immediately (fresh) compared to those processed after cryopreservation. However, the relative abundance of CD4 T cells expressing CCR5 (G), CD38 (H), and CXCR3 (I) was significantly increased after cryopreservation. Wilcoxon matched-pairs sign rank test were used for all analyses (n = 8 pairs for all except for CCR4 where n = 7, Wilcoxon matched-pairs signed rank tests were used for both analyses (n = 8), *denotes p-value <0.05, ns (non-significant) denotes p-value >0.06, exact p-value listed when 0.06>p>0.05).
Due to their importance in HIV entry, we also examined if the per cell expression level of CD4 and CCR5 (i.e., Median Fluorescence Intensity, MFI) were altered by cryopreservation. We observed no change in CD4 MFI (Fig 5A); however, we did observe a non-significant increase in CCR5 MFI after cryopreservation (MFI 4013 vs. 5434, p = 0.0547).
MFI of (A) CD4+ or (B) CCR5+ cells was compared between tissues processed immediately (fresh) compared to those processed after cryopreservation. Wilcoxon matched-pairs sign rank test was used for this analysis (n = 8, *denotes p-value <0.05, ns (non-significant) denotes p-value >0.06, exact p-value listed when 0.06>p>0.05.
Discussion
To our knowledge, this is the first in-depth characterization of cryopreservation on foreskin tissue. The number of live T cells that can be isolated per cm2 of foreskin tissue was significantly reduced by cryopreservation. However, despite overall cell loss, adequate numbers were recovered for sub-gating on all 10 phenotypic markers in this study, and neither the relative abundance of T cells expressing CD4 nor the expression level of CD4 (MFI) differed significantly between matched fresh and cryopreserved samples. Among CD4 T cells, the relative abundance of cells expressing CCR4, CCR6, CCR10, CD25, CD127, and HLA-DR did not differ significantly between matched fresh and cryopreserved samples. In contrast, the relative abundance of CD4 T cells expressing CD38, CCR5, and CXCR3 was significantly increased in cells isolated from foreskin tissue samples that had been cryopreserved. We are unable to discern if this increase is due to selective death of cell populations that do not express these markers, or if these receptors are upregulated in response to cryopreservation [16]. However, our data suggest that the per cell level of CCR5 expression (MFI) may also increase with cryopreservation, although with the relatively small sample size this increase did not reach statistical significance (p = 0.0547). Despite the increased relative abundance of CD38, CCR5, and CXCR3, the rankings of matched participant samples for fresh vs. cryopreserved were largely maintained. Therefore, while cryopreservation will lead to an overestimation of the expression of CD38, CCR5, and CXCR3, the gross ranking of participants based on the expression of these markers will be maintained. This suggests that cryopreserved foreskin samples can still be used to compare these markers across study groups, so long as all samples being analyzed are cryopreserved.
The increased relative abundance of cells expressing CCR5 post-cryopreservation contrasts with a previous report in PBMCs. Costantini et al. performed an in-depth characterization of the effect of cryopreservation on PBMCs isolated from both HIV-infected and HIV-uninfected individuals, and observed a marked decrease in the abundance of cells expressing CCR5+ post-cryopreservation [35]. It is possible that cryopreservation of the tissue as a whole, as opposed to a cell suspension, followed by enzymatic digestion may account for the different findings in foreskin and PBMCs. Foreskin tissues were cryopreserved intact, as a previous study found that the cryopreservation of cervicovaginal and colorectal as tissue and then isolating cells upon thawing yielded a greater number of cells than cryopreservation as a single-cell suspension [15]. Like our study, they found that CD4 expression did not significantly differ between matched fresh and cryopreserved tissue samples. While that study did not explore most of the phenotypic markers we used, they defined T cells and other white blood cells using functional markers such as CD3, CD4, CD8, CD66b, CD33, CD13, and CD45. Ultimately, they report that T cell functions (i.e., cytokine production in response to stimulation) remained similar after cryopreservation. A limitation of this study is that tissues were maintained in liquid nitrogen for a relatively short time (2–5 days), and it is unknown if longer cryopreservation may induce further changes in cell populations. However, previous studies in PBMCs [36] have demonstrated that cell populations are not affected by the duration of cryopreservation, but instead by the number of freeze/thaw cycles that a sample is exposed to.
Despite the substantial loss of overall cell numbers, our results indicate that many CD4 T cells phenotypic markers relevant to HIV susceptibility are unchanged by cryopreservation. Three markers investigated (CD38, CXCR3, and CCR5) were consistently higher after cryopreservation, but the relative ranking of participants was generally maintained. Therefore, it may be possible to use cryopreserved tissues to examine differences between participants, but these data caution against comparing participants whose tissues were not cryopreserved.
Supporting information
S1 Checklist. Inclusivity in global research questionnaire.
https://doi.org/10.1371/journal.pone.0297884.s001
(DOCX)
S1 Data. Raw data files for foreskin cryopreservation.
https://doi.org/10.1371/journal.pone.0297884.s002
(XLSX)
S1 Fig. Effect of tissue cryopreservation on the expression of phenotype markers in CD3 T cells.
https://doi.org/10.1371/journal.pone.0297884.s003
(TIF)
S1 Table. Details of antibodies used for flow cytometry.
https://doi.org/10.1371/journal.pone.0297884.s004
(TIF)
References
- 1. Gray RH, Kigozi G, Serwadda D, Makumbi F, Watya S, Nalugoda F, et al. Male circumcision for HIV prevention in men in Rakai, Uganda: a randomised trial. The Lancet. 2007;369: 657–666. pmid:17321311
- 2. Westercamp M, Bailey RC, Bukusi EA, Montandon M, Kwena Z, Cohen CR. Male Circumcision in the General Population of Kisumu, Kenya: Beliefs about Protection, Risk Behaviors, HIV, and STIs. PLOS ONE. 2010;5: e15552. pmid:21179493
- 3. Nagelkerke NJ, Moses S, de Vlas SJ, Bailey RC. Modelling the public health impact of male circumcision for HIV prevention in high prevalence areas in Africa. BMC Infect Dis. 2007;7: 16. pmid:17355625
- 4. Prodger JL, Galiwango RM, Tobian AAR, Park D, Liu CM, Kaul R. How Does Voluntary Medical Male Circumcision Reduce HIV Risk? Curr HIV/AIDS Rep. 2022;19: 484–490. pmid:36308579
- 5. Drain PK, Halperin DT, Hughes JP, Klausner JD, Bailey RC. Male circumcision, religion, and infectious diseases: an ecologic analysis of 118 developing countries. BMC Infect Dis. 2006;6: 172. pmid:17137513
- 6. Lawal TA, Olapade-Olaopa EO. Circumcision and its effects in Africa. Transl Androl Urol. 2017;6: 149–157. pmid:28540221
- 7. Prodger JL, Kaul R. The biology of how circumcision reduces HIV susceptibility: broader implications for the prevention field. AIDS Res Ther. 2017;14: 49. pmid:28893286
- 8. Tobian AAR, Serwadda D, Quinn TC, Kigozi G, Gravitt PE, Laeyendecker O, et al. Male circumcision for the prevention of HSV-2 and HPV infections and syphilis. N Engl J Med. 2009;360: 1298–1309. pmid:19321868
- 9. Gray RH, Kigozi G, Serwadda D, Makumbi F, Nalugoda F, Watya S, et al. The effects of male circumcision on female partner’s genital tract symptoms and vaginal infections in a randomized trial in Rakai, Uganda. Am J Obstet Gynecol. 2009;200: 42.e1–42.e7. pmid:18976733
- 10. Prodger JL, Gray R, Kigozi G, Nalugoda F, Galiwango R, Hirbod T, et al. Foreskin T-cell subsets differ substantially from blood with respect to HIV co-receptor expression, inflammatory profile, and memory status. Mucosal Immunol. 2012;5: 121–128. pmid:22089029
- 11. Prodger JL, Ssemaganda A, Ssetaala A, Kitandwe PK, Muyanja E, Mpendo J, et al. Schistosoma mansoni Infection in Ugandan Men Is Associated with Increased Abundance and Function of HIV Target Cells in Blood, but Not the Foreskin: A Cross-sectional Study. PLoS Negl Trop Dis. 2015;9: e0004067. pmid:26335139
- 12. Gray CM, O’Hagan KL, Lorenzo-Redondo R, Olivier AJ, Amu S, Chigorimbo-Murefu N, et al. Impact of chemokine C-C ligand 27, foreskin anatomy and sexually transmitted infections on HIV-1 target cell availability in adolescent South African males. Mucosal Immunol. 2020;13: 118–127. pmid:31619762
- 13. Galiwango RM, Park DE, Huibner S, Onos A, Aziz M, Roach K, et al. Immune milieu and microbiome of the distal urethra in Ugandan men: impact of penile circumcision and implications for HIV susceptibility. Microbiome. 2022;10: 7. pmid:35042542
- 14. Galiwango RM, Yegorov S, Joag V, Prodger J, Shahabi K, Huibner S, et al. Characterization of CD4+ T cell subsets and HIV susceptibility in the inner and outer foreskin of Ugandan men. Am J Reprod Immunol N Y N 1989. 2019;82: e13143. pmid:31081958
- 15. Hughes SM, Ferre AL, Yandura SE, Shetler C, Baker CAR, Calienes F, et al. Cryopreservation of human mucosal tissues. PLOS ONE. 2018;13: e0200653. pmid:30059507
- 16. Ebert LM, McColl SR. Up-regulation of CCR5 and CCR6 on distinct subpopulations of antigen-activated CD4+ T lymphocytes. J Immunol Baltim Md 1950. 2002;168: 65–72. pmid:11751947
- 17. Pi C-H, Hornberger K, Dosa P, Hubel A. Understanding the freezing responses of T cells and other subsets of human peripheral blood mononuclear cells using DSMO-free cryoprotectants. Cytotherapy. 2020;22: 291–300. pmid:32220549
- 18. Prodger JL, Abraham AG, Tobian AAR, Park DE, Aziz M, Roach K, et al. Penile bacteria associated with HIV seroconversion, inflammation, and immune cells. American Society for Clinical Investigation; 22 Apr 2021 [cited 12 Dec 2022]. pmid:33884964
- 19. Prodger JL, Gray RH, Shannon B, Shahabi K, Kong X, Grabowski K, et al. Chemokine Levels in the Penile Coronal Sulcus Correlate with HIV-1 Acquisition and Are Reduced by Male Circumcision in Rakai, Uganda. PLoS Pathog. 2016;12: e1006025. pmid:27898732
- 20. Hirbod T, Kong X, Kigozi G, Ndyanabo A, Serwadda D, Prodger JL, et al. HIV acquisition is associated with increased antimicrobial peptides and reduced HIV neutralizing IgA in the foreskin prepuce of uncircumcised men. PLoS Pathog. 2014;10: e1004416. pmid:25275513
- 21. Prodger JL, Hirbod T, Kigozi G, Nalugoda F, Reynolds SJ, Galiwango R, et al. Immune correlates of HIV exposure without infection in foreskins of men from Rakai, Uganda. Mucosal Immunol. 2014;7: 634–644. pmid:24150258
- 22. Ganor Y, Zhou Z, Tudor D, Schmitt A, Vacher-Lavenu M-C, Gibault L, et al. Within 1 h, HIV-1 uses viral synapses to enter efficiently the inner, but not outer, foreskin mucosa and engages Langerhans-T cell conjugates. Mucosal Immunol. 2010;3: 506–522. pmid:20571487
- 23. Zhou Z, Barry de Longchamps N, Schmitt A, Zerbib M, Vacher-Lavenu M-C, Bomsel M, et al. HIV-1 efficient entry in inner foreskin is mediated by elevated CCL5/RANTES that recruits T cells and fuels conjugate formation with Langerhans cells. PLoS Pathog. 2011;7: e1002100. pmid:21738469
- 24. Zhou Z, Xu L, Sennepin A, Federici C, Ganor Y, Tudor D, et al. The HIV-1 viral synapse signals human foreskin keratinocytes to secrete thymic stromal lymphopoietin facilitating HIV-1 foreskin entry. Mucosal Immunol. 2018;11: 158–171. pmid:28443609
- 25. Dinh MH, Anderson MR, McRaven MD, Cianci GC, McCoombe SG, Kelley ZL, et al. Visualization of HIV-1 interactions with penile and foreskin epithelia: clues for female-to-male HIV transmission. PLoS Pathog. 2015;11: e1004729. pmid:25748093
- 26. Fahrbach KM, Barry SM, Anderson MR, Hope TJ. Enhanced cellular responses and environmental sampling within inner foreskin explants: implications for the foreskin’s role in HIV transmission. Mucosal Immunol. 2010;3: 410–418. pmid:20410876
- 27. Fischetti L, Barry SM, Hope TJ, Shattock RJ. HIV-1 infection of human penile explant tissue and protection by candidate microbicides. AIDS Lond Engl. 2009;23: 319–328. pmid:19114867
- 28. Mohammadi A, Bagherichimeh S, Choi Y, Fazel A, Tevlin E, Huibner S, et al. Insertive condom-protected and condomless vaginal sex both have a profound impact on the penile immune correlates of HIV susceptibility. PLoS Pathog. 2022;18: e1009948. pmid:34982799
- 29. Mousset CM, Hobo W, Woestenenk R, Preijers F, Dolstra H, van der Waart AB. Comprehensive Phenotyping of T Cells Using Flow Cytometry. Cytometry A. 2019;95: 647–654. pmid:30714682
- 30. Gosselin A, Monteiro P, Chomont N, Diaz-Griffero F, Said EA, Fonseca S, et al. Peripheral blood CCR4+CCR6+ and CXCR3+CCR6+CD4+ T cells are highly permissive to HIV-1 infection. J Immunol Baltim Md 1950. 2010;184: 1604–1616. pmid:20042588
- 31. Trifari S, Kaplan CD, Tran EH, Crellin NK, Spits H. Identification of a human helper T cell population that has abundant production of interleukin 22 and is distinct from T(H)-17, T(H)1 and T(H)2 cells. Nat Immunol. 2009;10: 864–871. pmid:19578368
- 32. Manuszak C, Brainard M, Thrash E, Hodi FS, Severgnini M. Standardized 11-color flow cytometry panel for the functional phenotyping of human T regulatory cells. J Biol Methods. 2020;7. pmid:32313815
- 33. Kestens L, Vanham G, Vereecken C, Vandenbruaene M, Vercauteren G, Colebunders RL, et al. Selective increase of activation antigens HLA-DR and CD38 on CD4+ CD45RO+ T lymphocytes during HIV-1 infection. Clin Exp Immunol. 1994;95: 436–441. pmid:7907956
- 34. Meditz AL, Haas MK, Folkvord JM, Melander K, Young R, McCarter M, et al. HLA-DR+ CD38+ CD4+ T Lymphocytes Have Elevated CCR5 Expression and Produce the Majority of R5-Tropic HIV-1 RNA In Vivo ▿. J Virol. 2011;85: 10189–10200. pmid:21813616
- 35. Costantini A, Mancini S, Giuliodoro S, Butini L, Regnery CM, Silvestri G, et al. Effects of cryopreservation on lymphocyte immunophenotype and function. J Immunol Methods. 2003;278: 145–155. pmid:12957403
- 36. Li Y, Mateu E, Díaz I. Impact of Cryopreservation on Viability, Phenotype, and Functionality of Porcine PBMC. Front Immunol. 2021;12. Available: https://www.frontiersin.org/articles/10.3389/fimmu.2021.765667 pmid:34912338